KR20210005945A - Dermal filler and its application - Google Patents

Abstract

The present disclosure relates to photoinitiated dermal fillers, hyaluronic acid-rh collagen double crosslinked dermal fillers and hyaluronic acid-rh collagen semi-interpenetrated networks, as well as methods of use thereof, each of which is plant- It contains derived human collagen.

Description

Dermal filler and its application

Disclosed herein are photoinitiated and double crosslinked dermal fillers, including plant-derived human collagen, and cellular growth promoting scaffolds, as well as methods of using dermal fillers for soft tissue strengthening in some cases.

Collagen is a major protein responsible for the structural integrity of vertebrates and many other multicellular organisms. Collagen contains the major components of connective tissue and is the most abundant protein in mammals, including approximately 30% of the proteins found in the body. Loss or deterioration of collagen can occur as a result of aging or injury (Olsen et al., Adv Drug Deliv Rev. 2003 Nov. 28; 55(12):1547-67).

One common aspect of aging is the development of lines, fine lines, or wrinkles. Treatments involving the use of tissue-extracted collagen have been used to reduce or eliminate wrinkles, fine lines, or wrinkles. Similar treatments have been used to reduce scarring.

Collagen is also a component of tendons. Tendinopathy, a common injury usually associated with exercise and physical activity, is associated with degeneration and disordered arrangement of collagen fibers in the tendons. Healing of the injured tendon requires the coordinated activity of certain cells and the prolonged presence of the associated growth factor (GF) in the vicinity of the injury. Tennopathy is currently the main reason for musculoskeletal disease (Kaux et al. (Jan. 2011) J. Sport. Sci. Med. January:238-253). Tennopathy refers to a number of painful conditions that develop in and around the tendons and ligaments, which tend to arise from an imbalance between pathological changes resulting from tendon overuse and the resulting regenerative response (Andres et al. (2008) Clin. Orthop. Relat. Res. 466:1539-1554]). Tennopathy is associated with degeneration and misarrangement of collagen (Maffulli et al. (2003) Clin. Sport. Med. 22:675-692), occasionally fibrous microtear, increased vascularity, and mild inflammation. (Khan et al. (1999) Sport. Med. 27(6):393-408)). Clinically, tendonopathy is characterized by the onset of tendon stiffness, activity-related pain, decreased functionality, and occasionally localized swelling (Kaux 2011; Andres 2008). Collagen fibers present non-identical, irregular crimping, loosening, and increased waviness instead of a normal, tight, parallel, bundle-like appearance (Mafulli 2003). As the population is more active at an older age, the incidence of tendon injuries is expected to rise within the coming decades. A wide variety of treatments for tendonopathy, including physical therapy, pharmacological therapy, and combinations thereof, are available, but clinical results are not satisfactory and recurrence of symptoms is common (Kaux 2011). Injection of autologous platelet-rich plasma (PRP) for the treatment of tendonpathies has received widespread interest in the past decades (Delong et al. (2016) Curr. Orthpaedic Pract. 22:514-523); [Kaux et al. (2012) Wound Repair Regen. 20:748-756]; [Yuan et al. (2013) Muscles. Ligaments Tendons J. 3(3):139-49]; [Di Matteo et al. (2015) Musculoskelet. Surg. 99(1):1- 9]). PRP is the plasma fraction of blood containing high concentrations of platelets. When injected into the injured area, platelets release various types of growth factors (GFs) that are thought to facilitate the healing process. Among PRP-related GFs, vascular-endothelial growth factor (VEGF), transforming beta growth factor (TGF-beta), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), and fibroblast growth factor (bFGF) , Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) have been reported (Delong 2016; Yuan 2013; Harrison et al. (2011) Am. J. Sports Med. 39(4):729-734). Many in vitro studies and in vivo models show that PRP treatment enhances collagen expression and extracellular matrix growth, stimulates angiogenesis, increases cell migration, differentiation and proliferation, and supports healing of tendon injuries ( Yuan 2013; Kajikawa et al. (2008) J. Cell. Physiol. 215(3):837-845; Zhang et al. (2010) Am. J. Sports Med. 38(12):2477-2486) . However, clear clinical evidence of the efficacy of PRP treatment is limited (Delong 2016; Yuan 2013; Moraes et al. (2014)).

Collagen serves as a dominant component and primary structure-mechanical determinant of most tissue extracellular matrix (ECM) [see, eg, Kadler K. Birth Defects Res C Embryo Today. 2004; 72:1-11]; [Kadler KE, Baldock C, Bella J, Boot-Handford RP. J Cell Sci. 2007; 120:1955-1958.]; [Kreger ST. Biopolymers. 2010 93(8): 690-707]. Tropocollagens are typically three left-handed helices of procollagen (usually two identical helices and a third separate helix) that are conjugated to form a right-handed triple-helix tropocollagen. ), resulting in the formation of fibrils.

The layout and most properties of native collagen are determined by the triple helix domains that make up more than 95% of the molecule. This domain is composed of three alpha chains, each of which contains approximately 1,000 amino acids wrapped in a rope-like manner to form a tight triple helix structure. The triple helix is wound in such a way that the peptide bonds connecting adjacent amino acids are buried inside the molecule, so that the collagen molecule is resistant to attack by proteases such as pepsin.

Type I collagen represents prototypical fibrillar collagen and is a major collagen type in most tissues including bones, tendons, skin, arteries and lungs. Type I collagen fibers provide high tensile strength and limited extensibility. The most abundant molecular type of type I collagen is a heterotrimer consisting of two different alpha chains [alpha 1(I)] ₂ and alpha 2(I) (Inkinen, Connective Tissue Formation in Wound Healing an Experimental Study. , Academic Dissertation, Sep. 2003. University of Helsinki, Faculty of Science, Department of Biosciences, Division of Biochemistry]).

In all fibrotic collagen molecules, three polypeptide chains are constructed from repetitive Gly-X-Y triplets, where X and Y can be any amino acid, but are often iminoic acid proline and hydroxyproline. Collagen is particularly rich in glycine, proline and hydroxyproline amino acid residues, and the protein sequence of collagen strands often has a repetitive amino acid sequence. Procollagen is modified by adding hydroxyl groups to propyl and lysine residues. These hydroxylation reactions are catalyzed respectively by prolyl-4-hydroxylase and lysyl-hydroxylase. Thereafter, the hydroxyl groups on the lysine moiety are glycosylated and a triple helix is subsequently formed.

An important characteristic of fibril-forming collagen is that it is synthesized as a precursor procollagen containing globular N-terminal and C-terminal renal propeptides. The biosynthesis of procollagen is a complex involving many different post-translational modifications, including proline and lysine hydroxylation, N-linked and O-linked glycosylation, and both intrachain disulfide-bond formation and interchain disulfide-bond formation. It's a process. The enzymes that carry out these modifications act in a coordinated manner, ensuring the folding and assembly of correctly aligned and thermally stable triple-helix molecules.

Triconstituent polypeptide chains are assembled within the rough endoplasmic reticulum (RER) to form procollagen. Since the polypeptide chain is co-translated across the endoplasmic reticulum (ER) membrane, prolyl-4-hydroxylase (P4H)-dependent hydroxylation of proline and lysine residues occurs within the Gly-XY repeat region. do. The stability of the final triple-helix structure of collagen is highly dependent on the P4H-mediated hydroxylation of the collagen chain. Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in the post-translational modification of collagen. They sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagen and are essential for their functional activity (Wang et al. (2002) Matrix Biology, 21(7): 559-566). A single human enzyme, lysyl hydroxylase 3 (LH3), can catalyze all three successive steps in hydroxylysine linked carbohydrate formation (Wang et al. (2002) Matrix Biology, 21(7): 559- 566]). Once the polypeptide chain is completely translocated into the lumen of the endoplasmic reticulum, then the three pro-alpha chains are bonded through their C-propeptide to form a trimeric molecule, wherein the Gly-XY repeat region is its C- It forms a nucleation point at the distal end, ensuring correct alignment of the chains. Thereafter, the Gly-X-Y region is folded in the C-to-N direction to form a triple helix (Khoshnoodi et al. (2006) J. Biol. Chem. 281:38117-38121).

Hydroxylation of the proline residue is required to ensure the stability of the triple helix at body temperature, and once formed, the triple helix no longer serves as a substrate for the hydrosylation enzyme, so there is no difference between polypeptide chain modification and triple-helix formation. Temporary relationships are crucial. C-propeptides (and to a lesser extent N-propeptides) keep these procollagens soluble during the passage of procollagens out of the cell (Bulleid et al. (2000) Biochem. Socy. Transact., 28(4) ): 350-353]). After or during the secretion of the procollagen molecule into the extracellular matrix, the propeptide is removed by the procollagen N-proteinase and C-proteinase, thereby allowing the spontaneous self-assembly of the collagen molecule into fibrils. Trigger (Hulmes, 2002, J. Struct. Biol. January-February; 137(1-2):2-10). Removal of propeptide by procollagen N-proteinase and C-proteinase is required to lower the solubility of procollagen by more than 10,000-fold and initiate self-assembly of collagen into fibers at 37°C. Do. Crucial to this assembly process is the short telopeptides, which are non-triple-helical residues of the N-terminal and C-terminal propeptides that remain after digestion with N/C proteinase. These peptides function to ensure correct covalent registration of collagen molecules within the fibrillar structure and lower the critical concentration of self-assembly through their crosslinkable aldehydes (Bulleid et al. (2000) Biochem. Socy. Transact., 28(4): 350-353]).

Native collagen is generally present in connective tissue as telopeptide-containing collagen molecules packed side by side in fibrillar form. Each longitudinal path consists of molecules arranged in an end-to-end arrangement with some staggered longitudinal space compared to the next successive lateral adjacent longitudinal path. In this way, gaps are created between the facing end regions of successive molecules in a given longitudinal path and are joined by staggered faces of the molecules in parallel, laterally adjacent, cellular paths.

The dispersion and solubilization of native animal collagen is a variety of proteolytic enzymes that disrupt intermolecular bonds and remove immunogenic non-helix telopeptides without affecting the basic, rigid triple-helix structure that imparts the desired characteristics of the collagen. (U.S. Patents 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods of making purified soluble collagen. See issue). The resulting soluble atelocollagen can subsequently be purified by repeated precipitation at low pH and high ionic strength, followed by washing and re-solubilization at low pH. However, soluble formulations are typically contaminated by crosslinked collagen chains that reduce the homogeneity of the protein formulation.

Collagen has been selected from several biocompatible materials for use in tissue repair to support structural integrity, induce cellular invasion and promote tissue regeneration, due to its unique features and diverse profile in human function. Of the five major collagen types, type I collagen is the most abundant form of collagen in the human body.

Type I collagen can self-assemble into a fibrillar hydrogel capable of supporting tissue cells through bioactive adhesion sites. The addition of a methacrylate group to collagen produces collagen methacrylate (CMA), which is more resistant to degradation (Gaudet et al. Biointerphases (2012) 7:25-33). Thiolation of collagen can improve adhesion and mucoadhesion properties, and affect the swelling ability (Duggan et al., Eur. J. Pharm. Biopharm. (April 2015) 91:75-81).

Collagen's unique properties have contributed to its use in regenerative medicine products. Collagen can be used in pharmaceuticals (haemostatic compresses, sponges, healing dressings), medicine (prosthetics such as heart plates, tendons and ligaments, skin substitutes, fillers), dentistry (gum implants/gum disease) and cosmetic (additives, It provides biomaterials with features necessary for countless applications including anti-wrinkle agents and microcontainers for fragrance ingredients. All of the above-mentioned market-made collagen-based products require a large amount of raw collagen material for their production.

Human and animal-derived collagen, such as cadava or animal sources (bovine, pig or horse), and collagen-based products have been used for application, injection, transplantation, and oral ingestion. Uses include pre-shaping into a desired shape for repair or partial replacement of damaged bone or cartilage structures, injection into damaged joints, and injection as a dermal filler.

The use of animal-derived collagen (including human-derived collagen) is problematic due to the possible risk of contamination by non-conventional infectious pathogens. While the risk raised by bacterial or viral contamination can be completely controlled, prions are less inhibitory and present significant health risks. These infectious pathogens, which appear to have protein-like properties, are degenerative animal encephalopathy (sheep trembling disease, bovine spongiform encephalopathy) and human encephalopathy (Creefelz-Jakob disease, Gerstmann-Stroisler It is involved in the development of the syndrome (Gerstmann-Straussler syndrome), and kuru disease. Other diseases (eg, acquired immunodeficiency syndrome [AIDS], hepatitis, rabies, some cancers) can also be transmitted to recipients. Formal control is difficult to perform due to the too long time before the onset of encephalopathy and some other diseases. (See, generally, Castrow et al. (1983) J. Am. Acad. Dermatol. 9(6):889-93]; Siegle et al. (1984) Arch. Dermatol. 120(2):183-187] ).

Moreover, in some patients, treatment with human or animal collagen triggers cellular or humoral immune responses, including allergies. In addition, the quality of collagen generally degrades with the age of the organism or feed cadaveric or may degrade the subject to other factors. In addition, the extraction process causes significant structural damage that worsens its biological and mechanical function (Stein et al. (2009) Biomacromolecules 10(9):2640-2645); Shilo et al. (2013) Tissue Eng. Part. A 19(13-14):1519-1526]; Shosyov et al. (2013) Tiss. Eng. Part A 19(13-14):1527).

Plants expressing collagen chains are known in the art (eg, WO 2005/035442; U.S. Patent No. 6,617,431; U.S. Publication No. 2002/0098578; U.S. Publication No. 2002/0142391; Merle et al. (2002). ) FEBS Letters 515: 114-118]; [Ruggiero et al. (Mar. 3, 2000) FEBS Lett. 469(1):132-6]). While these plants can be used to produce collagen as well as collagen chains, these chains are erroneously hydroxylated so that they self-assemble, resulting in inherently unstable collagen, whether in the planta or not. For example, although plants can synthesize hydroxyproline-containing proteins, prolyl hydroxylase responsible for the synthesis of hydroxyproline in plant cells exhibits a relatively loose substrate sequence specificity compared to mammalian P4H. , The production of collagen containing hydroxyproline only at the Y position of the Gly-XY triplet requires plant co-expression of collagen and P4H genes (Olsen et al. (2003) Adv. Drug Deliv. Rev. , 55(12): 1547-1567]).

Processing of animal-derived “insoluble collagen” with plant-derived proteases such as ficin and/or papain is also known in the art (US Pat. Nos. 4,597,762, 5,670,369, 5,316,942, 5,997,895. And 5,814,328).

Attempts to produce human collagen that depend on the hydroxylation machinery naturally present in plants resulted in poor collagen in proline hydroxylation (Merle et al. (2002) FEBS Letters 515: 114-118). . These collagens melt or lose their triple helical structure at temperatures below 30°C. Co-expression of collagen and prolyl-hydroxylase results in a stable hydroxylated collagen that is biologically suitable for application at body temperature (Merle et al. (2002) FEBS Letters 515: 114-118).

The hydroxylysine of human collagen expressed in tobacco forms less than 2% of the hydroxylysine found in bovine collagen (0.04% residues/1.88% residues). This suggests that the plant endogenous lysyl hydroxylase is unstable to sufficiently hydroxylate lysine in collagen.

Recent technological advances are the introduction of five human genes encoding heterotrimeric type I collagen into tobacco plants, thereby introducing naive human type I collagen (rh collagen) (COLLPLANT ^TM , Israel; also St. Louis, Missouri SIGMA-ALDRICH®) has resulted in the development of a system for purification (see, eg, Stein H. (2009) Biomacromolecules 10:2640-5); [Yaari et al. (2013) Tiss. Eng. Part A 19(13/14): 1502-1506]; [Willard et al. (2013) Tiss. Eng. Part A 19(13/14): 1507-1518]; [Shilo et al. (2013) Tiss. Eng. Part A 19(13/14): 1519-1526]; [Shoseyov et al. (2013) Tiss. Eng Part A 19:1527-1533]; And [See Shoseyov et al. (Jan./Feb. 2014) Bioengineered 5:1, 1-4]]. Proteins are purified to homogeneity through cost-effective industrial processes that utilize the unique properties of collagen. Reference is also made to WO 2006/035442, WO 2009/053985, and patents and patent applications derived therefrom, all of which are incorporated by reference in their entirety as set forth herein.

Compared to tissue-extracted collagen, which can be partially denatured and cell binding domains removed, plant-derived human collagen type I has a more consistent structure and a greater number of cell binding domains (Shoseyov et al., Jan. /Feb. 2014) Bioengineered 5:1, 1-4]; [Majumdar et al. (2015) J. Biomed. Mater. Res. Part B: Appl. Biomater. 104B: 300-307]). rh collagen can form a functional three-dimensional (3D) matrix and scaffolding, in which 3D objects are produced in a layerwise manner using a computer model of the object through 3D bio-printing. It is applied to production (AM). Moreover, rhcollagen generally lacks the immunogenicity and predisease problems of tissue-extracted collagen.

A method for producing collagen in plants is available by expressing at least one type of collagen alpha chain and allowing the accumulation of this chain in subcellular compartments without endogenous P4H activity (US Pat. No. 8,455,717), likewise proteases. A method of producing atelocollagen from non-animal cell-derived human telopeptide-comprising collagen through treatment with is available (US Pat. No. 8,759,487).

Type I collagen and rhcollagen are considered candidates for use as major components of building materials in 3D-bioprinting. Various types of scaffolding have been used in cosmetic and other reconstruction applications.

In addition, the use of dermal fillers for strengthening soft tissues, for example reducing wrinkles, has increased. One possible method of using dermal fillers involves injecting a polymerizable dermal filler material into a desired area, followed by contouring or shaping the filler into the desired conformation. Polymerization and crosslinking of the material by one of a variety of methods can transform monomers in the injected material to form polymers and chains, which can form networks and retain the desired shaped shape. There are many ways to form polymers and crosslink polymers. Other methods involve photo-reactive reagents and photo-induced reactions that generate reactive species in monomer solutions. See, for example, US Patent No. 9,795,711; US Patent No. 8,945,624; US Patent No. 6,352,710; And US Publication No. 2009/0324722, as well as Elisseeff et al. (March 1999) Proc. Natl. Acad. Sci. USA 96: 3104-3107.

However, at least some of these approaches continue to focus on tissue-derived collagen or non-collagen polymers (eg, poly(vinyl alcohol), hyaluronic acid, or polyethylene glycol). Moreover, the use of tissue-extracted collagen is limited due to its sensitivity to temperature and ionic strength, which drives spontaneous gel formation under physiological conditions at temperatures above 20°C [eg, PureCol, Advanced BioMatrix, Inc. Reference]. The typical temperature-dependent formation of the tissue extracted-collagen gel significantly impedes the correct flowability. Keeping collagen at low temperatures until application is a possible solution to this phenomenon, but implies serious technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not become gel-like under these conditions. However, gelatin lacks the genuine tissue and cell interactions of native collagen, so significant biological functions are lost. Moreover, the viscosity makes it more difficult to be injected under the dermis using a micro-gauge needle, and also makes it more difficult to diffuse and shape the gelatin into smaller cavities.

Therefore, there is a need for improved injectable dermal fillers with tunable rheological and mechanical properties, and methods and uses thereof, and would be highly desirable and advantageous.

In one aspect, a double crosslinked dermal filler is disclosed, wherein the double crosslinked dermal filler,

(a) plant-derived human collagen; And

(b) crosslinked hyaluronic acid

Wherein the plant-derived human collagen is crosslinked to crosslinked hyaluronic acid.

In a related aspect, the plant-derived human collagen is

(a) containing type 1 recombinant human collagen (rh collagen);

(b) the crosslinked hyaluronic acid comprises crosslinked hyaluronic acid and non-crosslinked hyaluronic acid;

(c) a combination of these.

In a related embodiment, the crosslinking agent linking the crosslinked hyaluronic acid is different from the crosslinking agent linking the plant-derived human collagen with the crosslinked hyaluronic acid;

The ratio of the cross-linked hyaluronic acid: the plant-derived human collagen ranges from 4:1 to 1:2; It is a combination of these. In a further related embodiment, the crosslinking agent for crosslinking hyaluronic acid and the crosslinking agent for crosslinking plant-derived human collagen are independently 1,4-butanediol diglycidyl ether (BBDE), 1-[3-(dimethylamino)propyl ]-3-ethylcarbodiimide metiodide (EDC), Ν,N'-dicyclohexylcarbodiimide (DCC), Ν,N'-diisopropylcarbodiimide (DIC) [other See possible crosslinking agents].

In one aspect of the present application, a method of preparing a double crosslinked dermal filler comprising plant-derived human collagen crosslinked to crosslinked hyaluronic acid is disclosed, the method comprising:

(a) crosslinking hyaluronic acid;

(b) neutralizing the crosslinked hyaluronic acid;

(c) neutralizing plant-derived human collagen;

(d) mixing the neutralized crosslinked hyaluronic acid with neutralized plant-derived human collagen;

(e) adding a lower molecular weight hyaluronic acid (MW HA);

(f) crosslinking a mix of crosslinked hyaluronic acid and plant-derived human collagen; And

(g) dialysis of a double cross-linked cross-linked hyaluronic acid-plant-derived human collagen dermal filler.

In a related embodiment, the plant-derived human collagen comprises type 1 recombinant human collagen (rh collagen); The crosslinking agent linking the crosslinked hyaluronic acid of step (a) is different from the crosslinking agent linking the plant-derived human collagen of step (e) with the crosslinked hyaluronic acid; It is a combination of these. In a related embodiment, the ratio of crosslinked hyaluronic acid: plant-derived human collagen comprises a range of 4:1 to 1:2; The crosslinking agent for crosslinking hyaluronic acid and the crosslinking agent for crosslinking plant-derived human collagen are independently 1,4-butanediol diglycidyl ether (BBDE), 1-[3-(dimethylamino)propyl]-3-ethylcar Bodyimide metiodide (EDC), Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,N'-diisopropylcarbodiimide (DIC), or combinations thereof.

Furthermore, in one aspect, a method of filling the tissue space under the epidermis is disclosed, the method comprising:

(a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution,

(i) crosslinkable, plant-derived human collagen;

(ii) Hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof , Polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or modified derivatives thereof, carboxymethylcellulose or modified derivatives thereof , Crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; And

(iii) comprising a photoinitiator; And

(b) inducing polymerization by applying light to the surface of the epidermis superficial to the space.

In a related embodiment, the polymerizable solution components are introduced into the tissue space independently at about the same place and at about the same time, wherein the crosslinkable, plant-derived human collagen and photoinitiator are the hyaluronic acid (HA) or a modified derivative thereof. Introduced together with and independently from the poly(vinyl alcohol) (PVA) or a modified derivative thereof, the polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, Polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or the above A modified derivative, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, is independently introduced into the tissue space at approximately the same time period. In another related aspect, the method further comprises molding or sculpting the polymerizable solution or components of the polymerizable solution into a desired configuration within the tissue space, wherein , The step is concurrent with or subsequent to the step of applying light.

In another related embodiment, the polymerizable solution components are introduced together as a mixture into the tissue space, wherein the crosslinkable, plant-derived human collagen and photoinitiator are the hyaluronic acid (HA) or a modified derivative thereof, or the poly( Vinyl alcohol) (PVA) or a modified derivative thereof, or the polyethylene glycol (PEG) or a modified derivative thereof, or the oxidized cellulose (OC) or a modified derivative thereof, or the polymethylmethacrylate (PMMA) Microspheres or modified derivatives thereof, or tricalcium phosphate (TCP) or modified derivatives thereof, or calcium hydroxylapatite (CaHA) or modified derivatives thereof, or carboxymethylcellulose or modified derivatives thereof It is introduced with a derivative, or the crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

In another related embodiment, the polymerizable solution components are introduced into the tissue space independently of each other, wherein the crosslinkable, plant-derived human collagen and photoinitiator are the hyaluronic acid (HA) or a modified derivative thereof, or the poly( Vinyl alcohol) (PVA) or a modified derivative thereof, or the polyethylene glycol (PEG) or a modified derivative thereof, or the oxidized cellulose (OC) or a modified derivative thereof, or the polymethylmethacrylate (PMMA) Microspheres or modified derivatives thereof, or tricalcium phosphate (TCP) or modified derivatives thereof, or calcium hydroxylapatite (CaHA) or modified derivatives thereof, or carboxymethylcellulose or modified derivatives thereof Derivatives, or the crystalline nanocellulose (CNC) or modified derivatives thereof, or a combination thereof, are introduced together and independently from them.

In another related aspect, after introduction into the tissue space, the method further comprises shaping or shaping the polymerizable solution or components of the polymerizable solution into a desired arrangement within the tissue space, wherein the step Is concurrent with or subsequent to the step of applying light.

In another related aspect, the method is non-therapeutic, and the shaping or shaping step reduces wrinkles, folds, fine lines, wrinkles or scars, or a combination thereof.

In another related aspect,

(a) the crosslinkable, plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rh collagen);

(b) the hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), Modified derivatives of calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include methacrylated derivatives or thiolated derivatives;

(c) the hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), Calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) is crosslinked hyaluronic acid (HA), crosslinked poly(vinyl alcohol) (PVA), crosslinked polyethylene glycol (PEG), crosslinked oxidized cellulose (OC). ), crosslinked polymethylmethacrylate (PMMA) microspheres, crosslinked tricalcium phosphate (TCP), crosslinked calcium hydroxylapatite (CaHA), crosslinked carboxymethylcellulose, or crosslinked crystalline nanocellulose (CNC);

(d) a combination of (a) and (b), or a combination of (a) and (c).

In a further related embodiment, when MA-rh collagen is selected and hyaluronic acid or a derivative thereof, or crosslinked hyaluronic acid is selected, the ratio of HA: MA-rh collagen is 1:1, 2:1, 3:1, 4 :1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In one aspect of the present application, a method of filling a tissue space under the epidermis is disclosed, the method comprising introducing a double crosslinked dermal filler into the tissue space, wherein the double crosslinked dermal filler comprises:

(a) plant-derived human collagen; And

(b) crosslinked hyaluronic acid (HA) or a modified crosslinked derivative thereof, crosslinked poly(vinyl alcohol) (PVA) or a modified crosslinked derivative thereof, crosslinked polyethylene glycol (PEG) or a modified crosslinked derivative thereof, crosslinked oxidized cellulose ( OC) or a modified crosslinked derivative thereof, a crosslinked polymethylmethacrylate (PMMA) microspheres or a modified crosslinked derivative thereof, a crosslinked tricalcium phosphate (TCP) or a modified crosslinked derivative thereof, a crosslinked calcium hydroxylapatite (CaHA) or A modified crosslinked derivative thereof, a crosslinked carboxymethylcellulose or a modified crosslinked derivative thereof, a crosslinked crystalline nanocellulose (CNC) or a modified crosslinked derivative thereof, or a combination thereof;

The plant-derived human collagen is crosslinked crosslinked hyaluronic acid (HA) or a modified crosslinked derivative thereof, crosslinked poly(vinyl alcohol) (PVA) or a modified crosslinked derivative thereof, crosslinked polyethylene glycol (PEG) or a modified crosslinked derivative thereof , Crosslinked oxidized cellulose (OC) or modified crosslinked derivative thereof, crosslinked polymethylmethacrylate (PMMA) microspheres or modified crosslinked derivative thereof, crosslinked tricalcium phosphate (TCP) or modified crosslinked derivative thereof, crosslinked calcium hydroxide It is crosslinked to loxylapatite (CaHA) or a modified crosslinked derivative thereof, a crosslinked carboxymethylcellulose or a modified crosslinked derivative thereof, a crosslinked crystalline nanocellulose (CNC) or a modified crosslinked derivative thereof.

In a related embodiment, the plant-derived human collagen is type 1 human recombinant collagen (rh collagen), or a MA or thiolated derivative thereof; Hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or modified derivatives of crystalline nanocellulose (CNC) include methacrylated derivatives or thiolated derivatives; It is a combination of these.

In another related embodiment, when the crosslinked HA is selected, the ratio of the crosslinked HA: the plant-derived human collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In a related aspect, the method is non-therapeutic and reduces wrinkles, folds, fine lines, wrinkles or scars, or combinations thereof.

In one aspect of the present application, a polymerizable solution or a non-polymerizable solution for use in tissue strengthening is disclosed, wherein:

(a) the polymerizable solution comprises crosslinkable, plant-derived human collagen and a photoinitiator to induce polymerization prior to or simultaneously with the application of visible light, or

(b) the non-polymerizable solution comprises a plant-derived human collagen and a double-crosslinked dermal filler comprising crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE or crosslinked OC, wherein the plant-derived human collagen is Crosslinked to the crosslinked hyaluronic acid, the crosslinked PVA, the crosslinked PGE or the crosslinked OC;

The use includes injecting the polymerizable solution or non-polymerizable solution into the tissue space under the epidermis, followed by shaping or shaping the polymerizable solution or non-polymerizable solution into a desired configuration, such as wrinkles, folds, fine lines, Reducing wrinkles or scars.

In a related embodiment, the crosslinkable, plant-derived human collagen is methacrylated or thiolated; The polymerizable solution may be hyaluronic acid (HA) or a modified derivative thereof or a photopolymerizable modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof or a photopolymerizable modified derivative thereof, polyethylene glycol (PEG) or a Modified derivatives or photopolymerizable modified derivatives thereof, polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof or photopolymerizable modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof or photopolymerizable modifications thereof Calcium hydroxylapatite (CaHA) or a modified derivative thereof or a photopolymerizable modified derivative thereof, carboxymethylcellulose or a modified derivative thereof or a photopolymerizable modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof Or a photopolymerizable modified derivative thereof, or a combination thereof, wherein optionally the derivative thereof is a methacrylated derivative or a thiolated derivative; Or combinations thereof.

In another related aspect, tissue strengthening is required as a result of any medical or dental (gum implant/gingival disease) condition. In a further related aspect, tissue strengthening reduces wrinkles, folds, fine lines, wrinkles or scars, or a combination thereof.

In one aspect of the present application, a method of inducing a cellular growth promoting scaffold in a tissue space under the epidermis is disclosed, the method comprising introducing a solution into the tissue space, the solution comprising:

(a) plant-derived human collagen; And

(b) at least one growth factor or a source thereof,

Here, the method promotes healing or replacement of collagen-containing tissue.

In a related embodiment, the plant-derived collagen comprises type 1 recombinant human collagen (rh collagen); The source of the at least one growth factor comprises plasma or serum-rich plasma; The collagen-comprising tissue comprises skin; It is a combination of these.

In another embodiment, the method is non-therapeutic, and the cellular growth promoting scaffold fills in the tissue space to reduce wrinkles, folds, fine lines, wrinkles or scars, or combinations thereof.

In one aspect of the present application, a solution for use in inducing a cellular growth promoting scaffold is disclosed, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof, wherein the use is Injecting the solution into the tissue space under the skin, the use of which is to promote healing or replacement due to degradation or injury of collagen-containing skin tissue.

In a related embodiment, the plant-derived collagen comprises type 1 recombinant human collagen (rh collagen); The source of the at least one growth factor comprises plasma or serum-rich plasma; The collagen-comprising tissue comprises skin; It is a combination of these.

In another related aspect, the rhcollagen comprises a methacrylate or thiol derivative thereof.

In a related embodiment, the solution used in the method is hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, Carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and a photoinitiator to induce polymerization prior to or concurrently with the application of visible light; Crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE, or crosslinked OC, wherein the plant-derived human collagen is crosslinked to crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE, or crosslinked OC.

In a related aspect, the method is non-therapeutic, and the cellular growth promoting scaffold fills in the tissue space to reduce wrinkles, folds, fine lines, wrinkles or scars, or combinations thereof.

Disclosed herein is a solution for use in inducing a cellular growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof, wherein the use comprises applying the solution to the skin Injecting into the tissue space below, the use of which is to promote healing or replacement due to degradation or injury of the collagen-comprising tissue.

In a related embodiment, the source of the at least one growth factor comprises plasma or serum-rich plasma; Plant-derived collagen comprises type 1 recombinant human collagen (rh collagen); The collagen-comprising tissue comprises skin; It is a combination of these.

In another related embodiment, the solution for use is hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, Carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and a photoinitiator to induce polymerization prior to or concurrently with the application of visible light; It further comprises crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE, or crosslinked OC, wherein the plant-derived human collagen is crosslinked to crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE, or crosslinked OC.

In one aspect of the present application, a method of filling a tissue space under the epidermis is disclosed, wherein the method includes (a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution is (i) crosslinkable, plant- Derived human collagen; And (ii) a photoinitiator; And inducing polymerization by applying light to the surface of the epidermis superficial to the space.

In a related embodiment, the polymerizable solution further comprises shaping or shaping the polymerizable solution into a desired arrangement within the tissue space, wherein the step is concurrent with or subsequent to the step of applying light. to be.

In another related aspect, the method is non-therapeutic, and the shaping or shaping step reduces wrinkles, folds, fine lines, wrinkles or scars, or a combination thereof.

In another related embodiment, the crosslinkable, plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rh collagen).

Other objects, features and advantages of the present invention will become apparent from the following detailed description and drawings.

1A-1D illustrate the construction of various expression cassettes and vectors previously used to transform test plants. All coding sequences synthesized as part of the study were optimized for expression in tobacco. 1A shows a cloning scheme of collagen type I alpha I chain or collagen type II alpha 2 chain into plant expression vectors according to some embodiments of the present invention; 1B shows the cloning scheme of the enzyme prolyl-4-hydroxylase (P4H) into a plant expression vector according to some embodiments of the present invention; 1C shows the cloning scheme of proteinase C or proteinase N into plant expression vectors according to some embodiments of the present invention; 1D shows the cloning scheme of lysyl hydroxylase 3 (LH3) into plant expression vectors according to some embodiments of the present invention.
2 illustrates various co-transformation approaches used previously. Each expression cassette is indicated by the short name of the coding sequence. The coding sequence is specified in Table 1 . Each co-transformation was performed with two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector carrying 1, 2 or 3 expression cassettes. The promoter and terminator are specified in Example 1.
3 is a previous multiplex PCR screening of transformants showing plants positive for collagen alpha 1 (324 bp fragment) or collagen alpha 2 (537 bp fragment) or both.
4 is a previous Western blot analysis of transgenic plants produced by co-transformation 2, 3 and 4. Total soluble protein was extracted from tobacco co-transformants #2, #3 and #4 and tested with anti-collagen I antibody (#AB745 from Chemicon Inc.). The size marker was #SM0671 from Fermentas Inc. WT is a wild type tobacco. Positive collagen bands are visible in plants that are PCR positive for collagen type I alpha 1 or alpha 2 or both. A positive control band of 500 ng collagen type I from human placenta (#CC050 from Chemicon Inc., extracted by pepsin digestion from human placenta) was about in total soluble protein (about 150 μg) in samples from transgenic plants. Represents 0.3%. The larger band at about 140 kDa in the human collagen sample is the procollagen with its C-propeptide as detectable by the anti-carboxy-terminal pro-peptide of collagen type I antibody (#MAB1913 from Chemicon Inc.) . The smaller band at about 120 kDa in the human collagen sample is collagen without propeptide. Proline rich proteins (including collagen) consistently migrate as bands with higher molecular mass than expected on polyacrylamide gels due to their unusual composition. Thus, a collagen chain without a propeptide having a molecular weight of about 95 kDa migrates as a band of about 120 kDa.
5 is a previous Western blot analysis of transgenic plants produced by co-transformation #8 (carrying an apoplast signal translatingly fused to a collagen chain). Total soluble protein was extracted from transgenic tobacco leaves and tested with anti-collagen I antibody (#AB745 from Chemicon Inc.). The positive collagen alpha 2 band is visible in plants 8-141. Collagen type I from human placenta (#CC050 from Chemicon Inc.) serves as a control.
6A and 6B illustrate collagen triple helix assembly and thermal stability as previously qualified by heat treatment and trypsin or pepsin digestion. In Figure 6a , total soluble protein from tobacco 2-9 (expressing only collagen alpha1 and not P4H) and 3-5 (expressing both collagen alpha 1+2 and human P4H alpha and beta subunits). Were heat treated (15 min at 38° C. or 43° C.) followed by trypsin digestion (20 min at room temperature [RT]) and tested with anti-collagen I antibody in the Western blot procedure. The positive control was a sample of 500 ng human collagen I+ total soluble protein of wt tobacco. In Figure 6b , the total soluble protein was extracted from transgenic tobacco 13-6 (expressing collagen I alpha 1 and alpha 2 chains, human P4H alpha and beta subunits and human LH3 indicated by arrows) and heat treatment (33 C, 38° C. or 42° C. for 20 minutes), cooled immediately on ice to prevent reassembly of the triple helix, incubated with pepsin for 30 minutes at room temperature (about 22° C.) followed by standard western blot procedure. Was tested with an anti-collagen I antibody (#AB745 from Chemicon Inc.). The positive control was a sample of 50 ng human collagen I (#CC050 from Chemicon Inc., extracted from human placenta by pepsin digestion), which was added to the total soluble protein extracted from wild type (wt, wt) tobacco.
7 illustrates a previous Northern blot analysis performed on wild-type tobacco. The blot was probed with tobacco P4H cDNA.
8 is a previous Western blot analysis of transgenic plants produced by co-transformation 2, 3 and 13. Total soluble protein was extracted from tobacco co-transformers and tested with anti-human P4H alpha and beta and anti-collagen I antibodies.
9 is a hybrid sarcoma vacuole targeted plant A (2-300 +20-279) grown under normal light therapy; And previous Western blot analysis of 13-652 vacuole targeted plants grown for 8 days in the dark (lane 1). All plants express exogenous col1, col2, P4H-alpha and P4H-beta, as well as LH3 (PCR confirmed).
10 shows the purified collagen derived from tobacco-leaf after digestion by trypsin. Collagen was purified from tobacco plant transgenic leaf line number 13-6 crushed in 100 mM Tris buffer, as detailed in the Materials and Methods section, centrifuged, proteolyzed, and precipitated in a high salt concentration filler. After resuspension, the collagen-containing pellet was washed, dialyzed and concentrated to the final product. This gel shows the Coomassie staining analysis of the collected collagen samples, where lanes 1 and 2 are collagen produced after digestion of procollagen with 300 mg/L trypsin. Propeptide-free pig-derived collagen (0.5 mg/ml) was loaded and proceeded as positive controls for collagen type 1 alpha 1 and alpha 2 chains.
11 shows the purified collagen derived from tobacco-leaf after digestion by trypsin at various concentrations. Collagen was extracted and purified as in FIG. 10 after digestion with 20 mg/L trypsin (lanes 1 to 7) or 30 mg/L (lanes 8 to 10). The product was separated on 10% SDS PAGE and analyzed with Coomassie staining solution. Propeptide-free pig-derived collagen (0.5 mg/ml) was loaded and proceeded as positive controls for collagen type 1 alpha 1 and alpha 2 chains.
12 shows purified collagen derived from tobacco-leaf after digestion by trypsin and pepsin. Collagen was extracted and purified as in FIG. 10 after digestion with 30 mg/L trypsin and 1 μg/200 ml pepsin (lanes 1 to 2). The product was separated on 10% SDS PAGE and analyzed with Coomassie staining solution. Propeptide-free pig-derived collagen (0.5 mg/ml) was loaded and proceeded as positive controls for collagen type 1 alpha 1 and alpha 2 chains.
13 shows the collagen chains obtained upon degradation of procollagen by Subtilisin or Bromelain. Collagen is purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and incubated for 3 or 6 hours, subtilisin (1-25 mg/L) or bromelain (1-25 mg/L). Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. The untreated supernatant collected after homogenization and centrifugation served as a collagen-free negative control (lanes 3-4sup). Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
14 shows the collagen chains obtained upon decomposition of procollagen by papain. Collagen was purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and proteolyzed to papain (1-25 mg/L) over an incubation period of 3 or 6 hours. . Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. The untreated supernatant collected after homogenization, centrifugation and incubation at 15° C. for 3 hours (lane 3) or 6 hours (lane 2) served as a collagen-free negative control. Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
Fig. 15 shows the collagen chains obtained upon digestion of procollagen by ficin or savinase. Collagen is purified from tobacco plant transgenic leaf line no. 13-361 crushed in 100 mM Tris buffer, centrifuged, and ficin (1-25 mg/L) or sabinase () over an incubation period of 3 or 6 hours. 1-25 mg/L). Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. The untreated supernatant collected before proteolysis served as a collagen-free negative control (lane 3). Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
FIG. 16 shows collagen chains obtained upon degradation of procollagen by Protamex or Alcalase. Collagen is purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and Protamex (1-25 mg/L) or egg over an incubation period of 3 hours or 6 hours. Proteolysis with colorase (1-25 mg/L). Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. The untreated supernatant collected before proteolysis served as a collagen-free negative control (lane 14). Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
FIG. 17 shows collagen chains obtained upon degradation of procollagen by Esperase or Neutrase. Collagen is purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and after an incubation period of 3 or 6 hours, Esperase (1-25 mg/L) or Neutrase (1-25 mg/L). Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
18 shows collagen chains obtained upon decomposition of procollagen by 8.0 L of Esperase or Alcalase. Collagen is purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and after an incubation period of 3 or 6 hours, Esperase (1-25 mg/L) or Neutrase (1-25 mg/L). Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. The untreated supernatant collected after homogenization, centrifugation and incubation at 15° C. for 3 hours (lane 3) or 6 hours (lane 2) without proteolytic enzyme served as a collagen-free negative control. Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
19 shows collagen chains obtained in various purification steps after decomposition of procollagen by picin. Collagen was purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and proteolyzed with ficin (5 mg/L) after a 3 hour incubation period at 15°C. Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. Samples collected after pulverization, centrifugation, and incubation of the supernatant and evacuation were loaded into lane 5. Lanes 6 to 14 show samples of ficin-treated collagen at different stages of the purification process: lane 6: samples after ficin incubation and centrifugation; Lane 7: after salt precipitation and resuspension in 0.5 M acetic acid; Lane 8: sample as in lane 7 with added centrifugation step; Lane 9: sample as in lane 8 after resuspension and centrifugation in 0.5 M acetic acid; Lane 10: Mature collagen after resuspension and dialysis in 10 mM HCl. Lane 11: Sample as in lane 10 with an additional filtration step; Lane 12: sample as in lane 11 with an additional 5X concentration step; Lane 13: Sample as in lane 11 with an additional 20X concentration step; Lane 14: Sample as in lane 13 with an additional 5X concentration step. Untreated procollagen samples (lanes 3-4) served as negative controls. Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
20 shows collagen chains obtained in various purification steps after decomposition of procollagen by ficin. Collagen was purified from tobacco plant transgenic leaf line number 13-361 crushed in 100 mM Tris buffer, centrifuged, and proteolyzed with ficin (5 mg/L) after a 3 hour incubation period at 15°C. Samples were separated on 10% SDS PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen I. Samples collected after pulverization, centrifugation, and incubation of the supernatant and evacuation were loaded into lane 5. Lanes 6 to 14 show samples of ficin-treated collagen at different stages of the purification process: lane 6: samples after ficin incubation and centrifugation; Lane 7: after salt precipitation and resuspension in 0.5 M acetic acid; Lane 8: sample as in lane 7 with added centrifugation step; Lane 9: sample as in lane 8 after resuspension and centrifugation in 0.5 M acetic acid; Lane 10: Mature collagen after resuspension and dialysis in 10 mM HCl. Lane 11: Sample as in lane 10 with an additional filtration step; Lane 12: sample as in lane 11 with an additional 5X concentration step; Lane 13: Sample as in lane 11 with an additional 20X concentration step; Lane 14: Sample as in lane 13 with an additional 5X concentration step. Untreated procollagen samples (lanes 3-4) served as negative controls. Propeptide-free pig-derived collagen (2.5 μg) served as a positive control for the alpha 1 and alpha 2 chains (lane 1).
21 shows the collagen content of the post-pisin treated samples at various stages of purification. Collagen-containing samples were collected at each extraction and purification step of the reactor size AMS-based purification procedure described in the Materials and Methods section. Samples were treated with ficin (5 mg/L, 15° C., 3 hours) to remove propeptide, separated on 10% SDS PAGE, and stained with Coomassie staining solution.
22 shows the optimization of procollagen cleavage by food-grade picin: optimization of picin concentration and reaction time. The AMS-pelted procollagen-expressing tobacco leaf extract was resuspended in extraction buffer and then incubated with increasing concentrations of food-grade ficin (5-15 mg/L). Thereafter, the reaction mixture was incubated at 15° C. for 1 to 3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted against alpha-1 and alpha-2 collagen chains with anti-collagen I. Procollagen bands are indicated by white arrows, while red arrows indicate cleaved collagen bands.
23A-23C show optimization of procollagen cleavage by drug-grade escapement: optimization of escapement concentration and reaction time. The AMS-pelletized procollagen-expressing tobacco leaf extract was resuspended in extraction buffer and then incubated with increasing concentrations of drug-grade picin (2.5-10 mg/L). Thereafter, the reaction mixture was incubated at 15° C. for 0.5 to 3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted against alpha-1 and alpha-2 collagen chains with anti-collagen I. Arrows indicate the procollagen band and the collagen band.
24A-24B show optimization of procollagen cleavage by drug-grade escapement: optimization of pH and salt concentration in reaction buffer. AMS-pelletized procollagen-expressing tobacco leaf extract is resuspended in extraction buffer containing 10 mg/L drug-grade picin with increasing NaCl concentration (0.5 M to 3 M) at various pH values (5.5 to 9.5). Became. Thereafter, the reaction mixture was incubated at 15° C. for 1 hour. Cleavage was terminated by centrifugation, and protein samples of both the resulting pellet and supernatant were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and alpha-1 and alpha-2 with anti-collagen I. Immunoblotted against collagen chains. Arrows indicate collagen bands.
Figure 25 shows the optimization of procollagen cleavage by drug-grade picin: optimization of EDTA and L-cysteine concentrations in reaction buffer. AMS-pelletized procollagen-expressing tobacco leaf extract contains various concentrations of L-cysteine (10 mM to 100 mM--concentration top panel) and EDTA (8 mM to 80 mM--concentration bottom panel). Resuspended in extraction buffer (pH 7.5). Thereafter, the samples were incubated with 1 mg/L drug-grade picin for 1 hour at 15°C. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted against alpha-1 and alpha-2 collagen chains with anti-collagen I.
26 shows effective procollagen digestion by recombinant trypsin at pH 7.5. The AMS-pelted procollagen-expressing tobacco leaf extract was resuspended in extraction buffer (pH 7.5) containing L-cysteine and EDTA. Thereafter, the samples were incubated with 30 mg/L to 100 mg/L recombinant trypsin for 1 to 3 hours at 15°C. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted against alpha-1 and alpha-2 collagen chains with anti-collagen I.
Figure 27 shows the viscosity (eta[η], cP) as a function of shear rate, solid line- 2.7 mg/mL bovine collagen in phosphate buffered saline (PBS), and dashed line 2.79 mg/mL rh collagenase in PBS. ▼: Measurement at 4°C, ▲: Measurement at 37°C.
Figure 28 shows viscosity as a function of shear rate, 3.4 mg/mL bovine collagen in FB. ▼: Measurement at 4°C, ▲: Measurement at 37°C.
29 shows the viscosity as a function of shear rate, 10 mg/mL rh collagen-MA in PBS. ▲: Measurement at 4°C, ▼: Measurement at 37°C.
Figure 30 shows the viscosity measurement of rh collagen-MA in DMEM with and without the addition of HA/HAMA.
31 shows the storage coefficient and loss coefficient and tan phase shift angle of rh collagen-MA formulations at different concentrations before (top graph) and after (bottom graph) photocrosslinking.
Figure 32 shows the G'and G" values at 37° C. recorded in a frequency sweep test and plotted at 1 Hz.
Figures 33A-33C provide flow charts for the processing of rh collagen and rh collagen methacrylate. 33A shows the upstream isolation and processing of procollagen and collagen ( steps A-H ). Figures 33b-33c show the two phases of downstream processing ( step I-step M and step N-step P and step Z, respectively ).
FIG. 34 is a 5 mg/ml rh collagen methacrylate (CollMA) (solid linear curve) collMA:PVAMA ratio of 5:1 (solid linear curve), 2:1 (dashed linear curve), and 1:2 (dotted linear curve). Black curve) and the viscosity (eta[η], cP) of 5 mg/ml collagen MA + polyvinyl alcohol methacrylate (PVMA) (light gray curve). The viscosity of 5 mg/ml rh collagen methacrylate is reported for comparison (black curve).
Figure 35 shows 5 mg/ml CollMA (solid black curve) and 5 mg/ml collagen MA + hyaluronic acid methacrylate at a collMA:HAMA ratio of 5:1 (solid linear curve) and 2:1 (dashed linear curve). (HAMA) (gray curve) shows the viscosity. The viscosity of 5 mg/ml rh collagen methacrylate is reported for comparison (solid black curve). These materials have not yet been crosslinked, but will crosslink after injection. Viscosity is representative of the injectability of a material. (HAMA-HA methacrylate; collagen MA (ColMA)-rh collagen methacrylate.)
Figure 36 shows 5 mg/ml CollMA (solid black curve) and 5 mg/ml at collMA:OC ratios of 5:1 (solid linear curve), 2:1 (dashed linear curve), and 1:2 (dotted linear curve). The viscosity of ml collagen MA + oxidized cellulose (OC) (gray curve) is shown. The viscosity of 5 mg/ml rh collagen methacrylate is reported for comparison (solid black curve).
37 provides a comparison of the data from FIGS. 33-35 . This is 5 mg/ml CollMA (solid black curve) and 5 mg/ml collagen MA + different at ratios of 5:1 (solid linear curve), 2:1 (dashed linear curve), and 1:2 (dotted linear curve). Shows the viscosity of the additive (light gray to dark gray curve as indicated in the figure). The viscosity of 5 mg/ml rh collagen methacrylate is reported for comparison (solid black curve).
Figure 38 shows a polymerized scaffold of rh collagen methacrylate (ColMA) + additive at a collMA: additive ratio of 2:1. ColMA alone was compared to ColMA in combination with polyvinyl alcohol methacrylate (PVMA), hyaluronic acid methacrylate (HAMA), or oxidized cellulose (OC). The solution was mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.1%), and irradiated with ultraviolet (uv) light (365 nm) for 20 seconds.
39 shows a viability study. Top graph: When cultured in the presence of GF released from rh collagen-PRP matrix (black), in the presence of GF released from activated PRP (grey) and cultured under starvation conditions (white), normal human fibroblasts (nHDF ) Is a comparison of viability (and proliferation). Data are the average of two different fibroblast proliferation assays performed with PRP extracted from two different donors. *Significant difference (p<0.0002). Bottom inset: microscopic image of nHDF cells grown under starvation condition (c) and proliferated in the presence of GF released from activated PRP (b) in the presence of GF released from rh collagen matrix (a) in combination with PRP. . Images were taken 7 days after the cells were inoculated.
Figure 40 shows the scaffold weight as a function of time (each point is the average of 6 scaffolds, 2 rats per time point, 3 injections per rat).
41A-41B show two studies using the subcutaneous rat model. In the subcutaneous rat model, (a) PDGF content as a function of time (b) VEGF content as a function of time. *Significant difference between rh collagen matrix combined with PRP and PRP alone (p<0.038) and significant difference between rh collagen matrix alone and PRP alone (p<0.004). **Significant difference between rh collagen matrix alone and PRP alone (p<0.021) and significant difference between rh collagen matrix alone and rh collagen matrix in combination with PRP (a), and rh collagen matrix in combination with PRP and It is a significant difference between PRP alone and between rhcollagen matrix in combination with PRP and rhcollagen matrix alone (p<0.007)(b).
Figure 42 shows the integral of the nominal PDGF and VEGF content in the injected matrix over 45 days (or 30 days) in a rat model.
43A-43C show histopathological scoring of the Achilles tendon in a rat model of tendonosis treated with PRP or rh collagen/PRP matrix. (a) mature fibrosis, (b) the presence of mononuclear inflammatory cells, and (c) the presence of immature granulated tissue.
Figure 44 shows crosslinked hyaluronic acid (HA) (black curve -□), crosslinked hyaluronic acid (HA) + monomeric collagen (▼), or crosslinked hyaluronic acid (HA) + fibrilized from a 32-gauge needle and 1 ml syringe. It shows a comparison of the expression force (Newton, N) required for injection of collagen (▲) (Becton Dickinson [BD], ref. 309628). Crosslinked HA + monomeric collagen and crosslinked HA + fibrilized collagen are semi-interpenetrating networks, where the collagen is not crosslinked to anything.
Figure 45 shows the expression power (Newton, N) required for injection of crosslinked hyaluronic acid (HA) (black) or double crosslinked network of crosslinked hyaluronic acid (HA) and collagen (gray) from a 32-gauge needle and 1 ml syringe. A comparison is shown (Becton Dickinson [BD], ref. 309628).
Figure 46 is the viscosity (eta[η]) of crosslinked hyaluronic acid (HA) (black), crosslinked hyaluronic acid (HA) + monomeric collagen (▼), or crosslinked hyaluronic acid (HA) + fibrilized collagen (▲), cP).
47 shows a comparison of the viscosity (eta[η], cP) of a double crosslinked network (gray) of crosslinked hyaluronic acid (HA) (black) or crosslinked hyaluronic acid (HA) and collagen.
48A to 48B are (a) a mouse patch placed on top of a methacrylated collagen (collMA/rh collagen MA) solution and (B) polymerized into the skin upon irradiation with a white light emitting diode (LED) torch through the skin. And integrated methacrylated collagen (collMA/rh collagen MA).
49 shows two examples of dermal filler components. On the left side is the scheme of a semi-interpenetrating dermal filler comprising crosslinked hyaluronic acid (HA) and rh collagen. On the right side is a scheme of a double crosslinked dermal filler comprising crosslinked hyaluronic acid and rh collagen, wherein the crosslinked HA is further crosslinked to the rhcollagen. Light gray bars indicate HA-crosslinker, black strands indicate HA, rh collagen is indicated as thin gray strands, and the second crosslinker that crosslinks crosslinked HA to rh collagen is indicated as black circles.
Figure 50 : Storage coefficients for various double crosslinked formulations measured using a HAAKE-RHEO STRESS 600 ^TM instrument (THERMO SCIENTIFIC ^TM ) using cone (1-degree) versus plate arrangement (C35/1) and A graph depicting the rheological measure of the loss factor is shown. Frequency sweep measurements were performed at a constant strain of 0.8% and a frequency ranging from 0.02 Hz to 100 Hz. Storage factor (solid line) and loss factor (dashed line) of a representative double crosslinked formulation (see Table 7) compared to commercially available dermal fillers (solid line and dashed line: solid black line-commercially available product; solid ▼- formulation 2; solid line □-formulation 2A; solid line upward pentagon- formulation 3; solid line downward pentagon-formulation 1A; solid line ○-formulation 1; dashed line ▲-commercially available product G''; dashed line ▼-formulation 2G''; dashed line □ -Formulation 2A-G''; broken line ○- Formulation 1G''; broken line downward pentagon- Formulation 1A G``; broken line upward pentagon-Formulation 3'').
FIG. 51 shows a graph showing a comparison of the storage and loss factors of the formulations reported in FIG. 50 at f=1 Hz. (Open bar G'[Pa]; gray bar G'' [Pa])
Figure 52 is a MULTI-TEST 1-i MECMESIN ^TM compression tester machine equipped with a 1 ml LUER-LOK ^TM syringe (BECTON-DICKINSON ^TM ) and a 30 G needle used for Formulations 2, 2A and 3 (Table 8). A graph showing the measured, injectable properties of selected double crosslinked formulations is shown. Commercially available dermal fillers are included for comparison to double crosslinked formulations. The expression power as a function of plunger displacement (12 mm/min) of a representative double crosslinked formulation was compared to commercially available dermal fillers. (Black ▲ commercially available dermal filler; gray □-formulation 3; gray upward pentagon-formulation 2; gray ▼-formulation 2A)
Figure 53 is a highly crosslinked hyaluronic acid (HA), rh collagen methacrylate (MA), and / or various combinations of rh collagen (dashed line) before photocuring with visible light and rh collagen (solid line) after photocuring (Table 10) Reference) is shown in comparison to a highly crosslinked HA (a triangle with a black intermittent line aside). Prior to photocuring, storage and loss factors were measured using a HAAKE-RHEO STRESS 600 ^™ instrument (THERMO SCIENTIFIC ^™ ) using cone (1-degree) versus plate arrangement (C35/1). Frequency sweep measurements were performed at a constant strain of 0.8% and a frequency ranging from 0.02 Hz to 100 Hz. After light curing (6 minutes of visible light irradiation with white LED flashlight), the storage and loss coefficients were made using a HAAKE-RHEO STRESS 600 ^TM instrument (THERMO SCIENTIFIC ^TM ) using a serrated plate to plate form (PP20). Was measured. Frequency sweep measurements were performed under a constant shear rate of 3 Pa, a constant normal load of 0.3 N at a frequency ranging from 0.02 Hz to 100 Hz. (Solid line ▲-after formulation 4; solid line ▼-formulation 5-after; solid line □-formulation 6-after; broken line upward pentagon-formulation 4-before; broken line downward pentagon-formulation 5 before; broken line ○-formulation 6-before ; Dashed-dotted black sideways triangle-highly crosslinked HA)
FIG. 54 shows a graph showing a comparison of the storage and loss factors before and after photocuring of formulations 4, 5 and 6, as well as non-curable, highly crosslinked HA in Table 10, at a frequency of F = 1 Hz. (Open bar G'[Pa]; gray bar G'' [Pa])
Figure 55 is a selected double crosslinked formulation, measured using a MULTI-TEST 1- i MECMESIN ^™ compression tester machine equipped with a 1 ml LUER-LOK ^™ syringe (BECTON-DICKINSON ^™ ) and a 30 G needle used for all samples. A graph showing the injectability of is shown. Expression power as a function of plunger displacement (12 mm/min) of a representative double crosslinked formulation was compared to the highly crosslinked HA. (Black ▲-Highly crosslinked HA; Gray ▼- Formulation 4; Gray □-Formulation 5; Gray ○-Formulation 6)
FIG. 56 shows representative histological images on day 7 after subcutaneous injection of Formulations 2, 2A and controls (commercially available dermal fillers) into the back of Sprague Dawley rats. In each case, arrows indicate an enhanced immune response in Formulations 2 and 2A (not severe), indicating the onset of tissue regeneration.
FIG. 57 presents histology scores on Day 7 of formulations 2, 2A and controls from the tissues analyzed in FIG. 56. (Black-Control; Light Gray Formulation 2; Dark Gray Formulation 2A)
58 shows the results of photocurable histology scoring of photocurable dermal fillers on days 7, 14 and 20. (Black-highly crosslinked HA of control; gray-Formulation 4 highly crosslinked HA and rhColMA)
Figure 59 presents fibrosis score results on days 7 and 14 after injection of Formulation 4 (grey-highly crosslinked HA + rhColMA) vs. control (black-highly crosslinked HA).

Disclosed herein are photoinitiated dermal fillers and double crosslinked dermal fillers, and cellular growth promoting scaffolds, and methods of using them for, for example, but not limited to, soft tissue strengthening.

Collagen-producing plants can be used to produce collagen as well as collagen chains, but these chains are erroneously hydroxylated so that they self-assemble, which, whether in the planta or not, is the plant-derived human collagen of the present application. In contrast to the inherently unstable collagen results.

To reduce the present polymerizable and double-crosslinked solutions, and methods of use to practice, practitioners ensure the correct hydroxylation of collagen chains to ensure the characteristics of human type I collagen (e.g., molecular structure, temperature stability, cellular A plant expression approach was devised that enables the in-planta production of collagen that closely mimics sex interactions.

In one aspect, disclosed herein is a method of filling a tissue space under the epidermis, the method comprising:

(a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution,

(i) crosslinkable, plant-derived human collagen; And

(ii) comprising a photoinitiator; And

(b) inducing polymerization by applying light to the surface of the epidermis superficial to the space.

In certain embodiments, the method further comprises shaping or shaping the polymerizable solution into the desired arrangement within the tissue space prior to, or concurrently with, applying light. In another specific embodiment, the shaping or shaping step reduces wrinkles, folds, fine lines, wrinkles or scars.

In yet another specific embodiment, the polymer solution is hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, or a filler comprising a combination thereof. In one specific embodiment, the isolated plant-derived human collagen is optionally, such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmetha It is formulated with acrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, crystalline nanocellulose (CNC), or combinations thereof.

Modified derivatives include, but are not limited to, photopolymerizable versions of, for example, HA, PVA, PEG, or OC. Modifications include, but are not limited to, methacrylation or thiolation. In yet another specific embodiment, the light source is selected from light emitting diodes (LEDs), lasers, xenon lamps, and the like.

In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein crosslinks itself only under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to thiolated rhcollagen under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to any MA/thiolated additive under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to methacrylated HA under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to thiolated HA under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to methacrylated PVA under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to thiolated PVA under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to methacrylated PEG under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to thiolated PEG under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to the methacrylated OC under lighting conditions. In some embodiments, the methacrylate rhcollagen in the formulations disclosed herein is crosslinked to thiolated OC under lighting conditions.

Those skilled in the art will appreciate that the photocurable formulation is actually semi-IPN before curing and becomes IPN (inter-transmitted network) after curing. IPNs can encompass two entangled networks, each one crosslinked to itself and not the other.

In some embodiments, the crosslinked agent does not reduce the final total amount of rhcollagen (with light), as the non-modified rhcollagen cannot be crosslinked under illumination and thus does not enhance the final stiffness. Includes the ratio of non-modified rh collagen to tune stiffness after crosslinking. Methacrylated HA can also be added to this final formulation.

In some embodiments, HA or MA-HA may be crosslinked to itself using a crosslinking agent, such as but not limited to BDDE, as described in Example 23. In some embodiments, the crosslinking agent that crosslinks HA or MA-HA comprises divinyl sulfone (DVS) or glutaraldehyde. In certain embodiments, the BDDE crosslinked HA or MA-HA is not further crosslinked to rh collagen or MA-rh collagen, creating what is called an interpenetrating network ( FIG. 49 left side ).

In yet another specific embodiment, the plant-derived collagen comprises rh collagen. In another specific embodiment, the plant-derived collagen is obtained from a genetically modified plant. In another specific embodiment, the genetically modified plant is a genetically modified plant selected from tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. In particular, genetically modified plants are tobacco plants.

In yet another specific embodiment, the genetically modified plant is the expression of at least one gene sequence of human deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-alpha, P4H-beta, and LH3. Includes gender sequence. In another specific embodiment, the plant-derived human collagen comprises at least one modified human collagen alpha-1 chain as set forth in SEQ ID NO: 3 and expressed in a genetically modified plant; And at least one modified human collagen alpha-2 chain as shown in SEQ ID NO: 6 and expressed in a genetically modified plant; The genetically modified plants further express exogenous prolyl-4-hydroxylase (P4H). In another specific embodiment, the method further comprises expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. In yet another specific embodiment, the human collagen alpha-1 chain is encoded by a sequence as set forth in SEQ ID NO: 1 . In another specific embodiment, the human collagen alpha-2 chain is encoded by a sequence as set forth in SEQ ID NO: 4 .

In yet another embodiment, the exogenous P4H is mammalian P4H. In particular, exogenous P4H is human P4H. In yet another embodiment, the method further comprises targeting human collagen alpha-1 to the vacuoles of the plant or genetically modified plant, and degrading it to ficin. In yet another embodiment, the method further comprises targeting human collagen alpha-2 to the vacuoles of the plant or genetically modified plant, and degrading it to ficin.

In yet another embodiment, the plant-derived human collagen is atelocollagen. In another embodiment, the plant-derived human collagen is derived from SEQ ID NO: 1 and SEQ ID NO: 4 It is an atelocollagen having an amino acid (AA) sequence. Atelocollagen is derived by enzymatic digestion of procollagen (eg, with ficin), which is the product of SEQ ID NO: 1 and SEQ ID NO: 4 .

In yet another embodiment, the photoinitiator reacts to visible light to induce polymerization of the polymerizable solution. In particular, visible light has a wavelength of 390 nm to 800 nm. In particular, the photoinitiator is selected from the group consisting of eosin Y+ triethanolamine, riboflavin, and the like.

In another embodiment, the photoinitiator induces polymerization of the polymerizable solution by reacting to ultraviolet (uv) light. In particular, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl Propan-1-one (IRGACURE® 2959).

In another embodiment, the photoinitiator reacts to infrared light to induce polymerization of the polymerizable solution.

In yet another embodiment, the polymeric solution is introduced into the tissue space through a hollow needle or cannula ranging from 27 gauge to 33 gauge.

In yet another embodiment, the polymeric solution in the tissue space is shaped or shaped into the desired configuration via manual massage. In another embodiment, the polymerizable solution in the tissue space is shaped or shaped into the desired configuration through a shaping or shaping mechanism.

In yet another embodiment, the polymerizable solution in the tissue space is essentially non-gelable at room temperature. In another embodiment, the polymerizable solution in the tissue space is essentially non-gelable at 37°C. In yet another embodiment, the polymerizable solution comprising plant-derived human collagen comprises tissue-extracted human or animal-derived collagen, such as but not limited to bovine, pig or horse collagen, at the same concentration and formulation. It has a reduced viscosity at room temperature compared to similar polymeric solutions. In another embodiment, the polymerizable solution comprising plant-derived human collagen exhibits a reduced viscosity at 37° C. compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. Have.

As used throughout, the term “animal-derived collagen” can encompass bovine, porcine or horse collagen or rat tail collagen and is in contrast to human-derived collagen.

In yet another embodiment, the polymerizable solution comprising plant-derived human collagen is at reduced force at room temperature compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. It is introduced into the tissue space. In yet another embodiment, the polymerizable solution comprising plant-derived human collagen has a reduced potency at 37° C. compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. Is introduced into the tissue space.

In another embodiment, disclosed herein is the use of a polymeric solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein the polymerizable solution is methacrylated or thiol A photoinitiator to induce polymerization prior to or concurrently with application of modified crosslinkable, plant-derived human collagen and visible light, and the polymerizable solution in the desired batch to reduce wrinkles, folds, fine lines, wrinkles, or scars. Shaped or shaped. In certain embodiments, the polymer solution is hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) Or a modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose Or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a filler comprising any combination thereof.

Modified derivatives include, but are not limited to, for example, HA, PVA, PEG, OC, PMMA, TCP, CaHA, carboxymethylcellulose, or photopolymerizable versions of CNC. Modifications include, but are not limited to, methacrylation or thiolation.

In another aspect, disclosed herein is a method of filling a tissue space under the epidermis, the method comprising:

(a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution comprises crosslinkable, plant-derived human collagen.

The present technology relates in part to cosmetic and medical collagen-based polymeric fillers that form polymerizable moldable compositions upon photoactivation using a light source, such as an engineered light source. Polymeric fillers include crosslinkable, plant-derived human collagen together with a photoinitiator.

The technology of interest typically has the advantage of enabling in -situ formation of custom contoured dermal fillers or implants without invasive surgical intervention or general anesthesia. In general, a collagen-based polymeric solution is introduced into the tissue space under the skin (i.e., under the epidermis), and the polymerization is applied to the skin surface, i.e. Induced by exposure.

The in-situ polymerization method provides a cosmetic and medical corrective and/or augmentation procedure using a polymeric solution containing a polymer component capable of forming an insoluble crosslinked crosslinkable network upon activation with a visible light source. do.

In some embodiments, the dermal fillers or cellular growth promoting scaffolds disclosed herein are for cosmetic use. In some embodiments, the dermal filler or cellular growth promoting scaffold disclosed herein is for medical orthodontic use. In some embodiments, the dermal fillers or cellular growth promoting scaffolds disclosed herein are for use in enhancement procedures such as, for example, but not limited to tissue strengthening. In some embodiments, the double crosslinked dermal filler disclosed herein is for cosmetic use. In some embodiments, the double crosslinked dermal filler disclosed herein is for medical orthodontic use. In some embodiments, the double crosslinked dermal filler disclosed herein is required as a result of a medical or dental (gum implant/gum disease) condition. In some embodiments, the double crosslinked dermal fillers disclosed herein are required as a result of a medical condition requiring skin strengthening. In some embodiments, the double crosslinked dermal fillers disclosed herein are for use in enhancement procedures such as, for example, but not limited to tissue strengthening. In some embodiments, the photocurable dermal filler disclosed herein is for cosmetic use. In some embodiments, the photocurable dermal filler disclosed herein is for medical orthodontic use. In some embodiments, photocurable dermal fillers disclosed herein are required as a result of a medical or dental (gum implant/gum disease) condition. In some embodiments, photocurable dermal fillers disclosed herein are required as a result of a medical condition requiring skin strengthening. In some embodiments, photocurable dermal fillers disclosed herein are for use in augmentation procedures such as, for example, but not limited to tissue strengthening. In some embodiments, the cellular growth promoting scaffolds disclosed herein are for cosmetic use. In some embodiments, the cellular growth promoting scaffold disclosed herein is for medical orthodontic use. In some embodiments, the cellular growth promoting scaffold dermal filler disclosed herein is required as a result of a medical or dental (gum implant/gingival disease) condition. In some embodiments, the cellular growth promoting scaffold dermal filler disclosed herein is required as a result of a medical condition requiring skin strengthening. In some embodiments, medical orthodontic uses include treating tendinitis. In some embodiments, the cellular growth promoting scaffolds disclosed herein are for use in enhancement procedures such as, for example, but not limited to tissue strengthening.

In some embodiments, tissue strengthening is strengthening of skin tissue.

In some embodiments, the use of a dermal filler comprising a cellular growth promoting scaffold disclosed herein is in humans. In some embodiments, the use of a dermal filler comprising a cellular growth promoting scaffold disclosed herein in a human reduces wrinkles, folds, fine lines, wrinkles, or scars, or any combination thereof. In some embodiments, the reduction of wrinkles, folds, fine lines, wrinkles, or scars, or any combination thereof, is for cosmetic purposes. In some embodiments, the reduction of wrinkles, folds, fine lines, wrinkles, or scars, or any combination thereof, is for cosmetic purposes. In some embodiments, the use of a dermal filler comprising a cellular growth promoting scaffold disclosed herein in humans strengthens a tissue, such as, but not limited to, epidermal or dermal tissue. In some embodiments, the tissue strengthening is for cosmetic purposes. In some embodiments, tissue strengthening is for medical treatment. In some embodiments, tissue strengthening is part of an augmentation procedure. In some embodiments, tissue strengthening is part of a skin enhancement procedure.

In some embodiments, tissue strengthening is required as a result of any medical or dental (gum disease/gingival implant) condition.

In certain embodiments, dermal fillers for use as described herein are interpenetrated (IPN) networks or semi-interpenetrated (semi-IPN) networks in which different components may be crosslinked to themselves but not each other. Includes network. In some embodiments, the IPN or semi-IPN dermal filler is rh collagen and a filler such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or derivatives thereof. , Or a combination thereof. In some embodiments, the IPN or semi-IPN comprises rh collagen and crosslinked HA. In some embodiments, the IPN or semi-IPN is a rh collagen derivative, e.g., but not limited to a methacrylated rh collagen or a derivative of thiol rh collagen and/or a filler, e.g., but not limited to methacrylated. HA, PVA, PEG, or OC, or thiolated HA, PVA, PEG, or OC, or combinations thereof.

In some embodiments, the IPN or semi-IPN network or double-crosslinked network comprising a dermal filler has a filler, such as, but not limited to, a ratio of HA, PVA, PEG, or OC: rh collagen to 1:1, 2: 1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network comprising the dermal filler is a MA-filler, such as, but not limited to, HA, PVA, PEG, or OC: rh collagen ratio of 1:1, 2:1, 3 Included as :1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network comprising a dermal filler has a filler, such as, but not limited to, a ratio of HA, PVA, PEG, or OC: MA-rh collagen to 1:1, 2:1, 3 Included as :1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network comprising the dermal filler has a ratio of MA-filler: MA-rh collagen to 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In some embodiments, the IPN or semi-IPN or double crosslinked filler comprising a dermal filler has a ratio of HA:rh collagen to 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN or double crosslinked filler comprising a dermal filler has a ratio of MA-HA:rh collagen to 1:1, 2:1, 3:1, 4:1, 5:1, Include 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network comprising the dermal filler has a HA: MA-rh collagen ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, Include 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network comprising the dermal filler has a ratio of MA-HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In some embodiments, the IPN or semi-IPN or double crosslinked filler comprising a dermal filler has a filler: rh collagen ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN or double-crosslinked filler comprising a dermal filler has a ratio of MA-HA, or MA-PVA, or MA-PEG, or MA-OC:rh collagen, of 1:1, 2 Included as :1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN comprising the dermal filler has a ratio of MA-HA, or MA-PVA, or MA-PEG, or MA-OC: MA-rh collagen, of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN comprising the dermal filler has a ratio of MA-HA, or MA-PVA, or MA-PEG, or MA-OC: MA-rh collagen, of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In some embodiments, the IPN or semi-IPN network comprising a dermal filler or a double crosslinked dermal filler comprises a cellular growth promoting scaffold.

In certain embodiments, the dermal filler for use described herein is a photocurable dermis comprising at least one component, such as, but not limited to, a methacrylate-rh collagen derivative or a thiol-rh collagen derivative. Contains fillers. In some embodiments, the curable dermal filler is rh collagen and a filler such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or derivatives thereof, or Includes a combination. In some embodiments, the photocurable dermal filler comprises MA-rh collagen and HA or derivatives thereof. In some embodiments, the photocurable dermal filler is a rh collagen derivative, such as, but not limited to, a methacrylated rh collagen or a derivative of thiol rh collagen and/or a filler, such as, but not limited to, methacrylated HA. , PVA, PEG, or OC, or thiolated HA, PVA, PEG, or OC, or combinations thereof.

In some embodiments, the photocurable dermal filler is a filler, such as, but not limited to, HA, PVA, PEG, or OC, or a derivative thereof: rh collagen ratio of 1:1, 2:1, 3:1, 4: 1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler is a filler, such as, but not limited to, HA, PVA, PEG, or OC, or a derivative thereof: MA-rh collagen in a ratio of 1:1, 2:1, 3:1, Includes 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler is a filler, such as, but not limited to, a ratio of HA, PVA, PEG, or OC: thiol-rh collagen to 1:1, 2:1, 3:1, 4:1, It includes 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler has a ratio of MA-filler: MA-rh collagen to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1 Included as :3, 1:4, 1:5, 1:6, or 0:1.

In some embodiments, the photocurable dermal filler has a ratio of HA: MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3. , 1:4, 1:5, 1:6, or 0:1, 1:3, 1:4, 1:5, or 0:1. In some embodiments, the photocurable dermal filler has a ratio of MA-HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1 Included as :3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler has a ratio of PVA, PEG, or OC: MA-rh collagen to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1: 2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler comprises MA-PVA, MA-HA-, or OC: MA-rh collagen 1:1, 2:1, 3:1, 4:1, 5:1, 6:1 , 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the HA component of the photocurable dermal filler comprises crosslinked HA or crosslinked MA-HA.

Throughout this application, various embodiments of dermal fillers and their uses may be presented in a range format. It is to be understood that the description of the range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, descriptions of ranges such as 1:1 to 6:1 include subranges such as 1.1:1, 1.2:1, 1.3:1 to 5.9:1, 1:1.1 to 1:1.9, and the like, as well as corresponding ranges and Individual numbers within its fraction, for example 1, 2, 3, 4, 5 and 6, should be considered specifically disclosed. This applies regardless of the width of the range.

Whenever a numerical range is presented herein, it is meant to include any recited numerical value (fraction or integer) within the stated range. The phrases "range/range" between the first presented number and the second presented number "between" and the first presented number "to" and the "range/range" of the second presented number are used interchangeably herein, and the first presented number It is meant to include the number and the second presented number and all fractions and integers in between.

For example, the present disclosure relates to crosslinkable, plant-derived human collagen alone or as fillers such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC) , Or in combination with a combination thereof, provides a dermal filler for the tissue space under the epidermis, which is crosslinked to form a water-insoluble, crosslinked polymer formulation in drilling upon activation of visible light in the presence of a photoinitiator. Fillers can be provided. In some embodiments, the collagen is methacrylated or thiolated.

In some embodiments, dermal fillers provided for the uses described herein form an IPN or semi-IPN network. In some embodiments, dermal fillers provided for the uses described herein form a double crosslinked network.

In certain embodiments, double crosslinked dermal fillers provided for the uses described herein are crosslinked fillers such as crosslinked hyaluronic acid (HA), crosslinked poly(vinyl alcohol) (PVA), crosslinked polyethylene glycol (PEG), or crosslinked Oxidized cellulose (OC), or a cross-linked derivative thereof, or a combination thereof, cross-linked rh collagen. In certain embodiments, the double crosslinked dermal filler provided for the uses described herein is rh collagen further crosslinked to a methacrylated-crosslinked or thiolated-crosslinked filler such as HA, PVA, PEG, or OC. Includes.

In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked filler: rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of MA-filler to rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked filler: MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1: 2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of MA-filler: MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1: 2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of thiolated-filler: rhcollagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1: 2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked filler: thiolated-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of thiolated-filler: thiolated-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In certain embodiments, in the double crosslinked dermal filler, crosslinked HA: crosslinked MA-HA is further crosslinked to rhcollagen or methacrylated rhcollagen or thiol rhcollagen resulting in a double crosslinked dermal filler. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked HA: rh collagen or methylated rh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1 , 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked MA-HA: rh collagen or methylated rh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4:1, 5 :1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked MA-HA: rh collagen or methylated rh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4:1, 5 :1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In certain embodiments, in a double crosslinked dermal filler, crosslinked PVA, PEG, or OC, or crosslinked MA-PVA, MA-PEG, or MA-OC is further crosslinked to rh collagen or methacrylated rh collagen. Resulting in a double crosslinked dermal filler. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked PVA, PEG, or OC: rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked MA-PVA, MA-PEG, or MA-OC:rh collagen is 1:1, 2:1, 3:1, 4:1, 5: 1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked PVA, PEG, or OC: MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6: 1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked MA-PVA, MA-PEG, or MA-OC: MArh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4: 1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In certain embodiments, in the double crosslinked dermal filler, the crosslinked thiol-PVA, thiol-PEG, or thiol-OC is further crosslinked to rh collagen or methacrylated rh collagen resulting in a double crosslinked dermal filler. do. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked thiol-PVA, thiol-PEG, or thiol-OC:rh collagen is 1:1, 2:1, 3:1, 4:1, 5 :1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in the double crosslinked dermal filler, the ratio of crosslinked thiol-PVA, thiol-PEG, or thiol-OC: MArh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4 :1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

In some embodiments, any water soluble coupling agent capable of crosslinking hyaluronic acid to collagen may be used. Some non-limiting examples of coupling agents include carbodiimides such as Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,N'-diisopropylcarbodiimide (DIC), or 1-ethyl-3 -(3-dimethylaminopropyl)carbodiimide (EDC), and the like. The carbodiimide coupling agent can facilitate ester or amide bond formation without becoming part of the linkage. In other words, the ester linkage or the amide linkage may contain an atom from a carboxylate group from one of hyaluronic acid or collagen, and a hydroxyl group or an amine group from the others. However, other coupling agents that become part of the crosslinking group may be used. The concentration of the coupling agent can vary. In some embodiments, the coupling agent can be present at about 2 mM to about 150 mM, about 2 mM to about 50 mM, about 20 mM to about 100 mM, or about 50 mM. In some embodiments, the coupling agent is an EDC present at a concentration of about 20 mM to about 100 mM, about 2 mM to about 50 mM, or about 50 mM. In some embodiments, the coupling agent is an EDC present in an amount of EDC equal to 10-fold to 100-fold the number of free amines in rhcollagen. In some embodiments, the coupling agent is an EDC present in an amount of EDC equal to 50-fold the number of free amines in rh collagen. Increasing the carbodiimide concentration below about 50 mM may result in a crosslinked macromolecular matrix with greater hydrogel stiffness and/or less swelling.

Those of skill in the art will understand that dermal fillers comprising double crosslinks, which are crosslinked to themselves and then also crosslinked to rh collagen, are distinct from dermal fillers comprising direct crosslinking of collagen and HA using a single type of crosslinking agent in a single reaction. will be. The properties of these dermal fillers are different.

As an example, the present polymerizable solution can be used to block or fill various lumens and voids just below the skin surface. Therefore, the present technology provides a method of augmenting tissue in a host, such as a human patient, wherein the polymeric solution of interest is applied to a tissue site in need of augmentation or by using a method known in the art. It is injected into the tissue site and, once applied, is introduced to the site of interest by exposing the overlying body surface to visible light to cause polymerization of the deposited polymeric solution.

By “reinforcing” is meant to repair, prevent or mitigate defects, particularly defects due to the loss or absence of such tissue, by providing, enhancing or replacing with a polymer or network of interest to the tissue. Enhancement can also be used to supplement the natural structure or trait, i.e., for example, to increase the size of existing body parts, such as lips, nose, breast, ears, parts of organs, chin, cheeks, etc. It is meant to include additional construction to. Thus, tissue strengthening may include filling or reduction of, for example, wrinkles, folds, wrinkles, scars, minor facial depressions, cleft lip, superficial wrinkles in or on the face, neck, hands, feet, fingers, and toes; Correction of minor deformations due to aging or disease, including hands and feet, fingers and toes; Strengthening of the vocal cords or gates to restore speech; Dermal filling of sleep lines and expression lines; Replacement of dermal and skin tissue loss due to aging; Strengthening the lips; Filling of the wrinkles around the eye and the orbital groove; Breast strengthening; Chin strengthening; Strengthening of the cheek and/or nose; Filling of soft tissue, skin or indentation in the skin, for example due to excessive liposuction or other trauma; Filling of acne or traumatic scars and rhytids; It may include filling of the nasolabial line, the nasoglabellar line, and the intraoral line.

In some embodiments, the polymeric solution of interest has a viscosity suitable for preparation for extrusion through a delivery means, such as a microsurgical needle (e.g., a needle having at least 27 gauge, at least 33 gauge or finer gauge) at the temperature of use. It encompasses a polymerizable solution having. Therefore, a solution that is "injectable" has a texture and viscosity that allows flow through a suitable delivery device, such as a surgical needle, other surgical instrument, or other means of delivery, such as equipment used in endoscopic or percutaneous disc dissection procedures. It is a solution. Therefore, the polymeric solution of interest is injectable through a suitable applicator as known in the art, such as catheters, cannulas, needles, syringes, tubular instruments, and the like.

Once injected into the tissue space, the polymerizable solution can be manipulated, massaged, shaped or shaped into the desired contour within the tissue space, typically after the photoinitiation of polymerization has been triggered. In one embodiment, such manipulation, massage, shaping or shaping is performed during the gelling process. Polymerisable, polymerized, or partially polymerized solutions can be applied to, for example, shaping means, such as surgical depressors or other tools or devices, fingers, palms, finger joints, etc. with a flat or curved surface. Can be used to shape by external manipulation.

Surprisingly, the genetically modified, crosslinkable, plant-derived human collagen of the present method enables the use of improved collagen-containing dermal fillers, and smaller gauge needles and reduced injection power, as well as smaller tissue space. It provides a method of filling the dermis improved by its ability to fill.

The "expression force" of an injection (Newton, N) includes the force required for injection from a needle or cannula.

“Absolute Viscosity” (“Kinematic Viscosity”) is the resistance of a fluid to flow when a force is applied. Absolute viscosity is proportional to the ratio of force to velocity. The Greek letter η (eta) denotes the absolute viscosity in the calculation. Absolute viscosity is commonly measured in cP because many common fluids have viscosities between 0.5 cP and 1,000 cP.

A “gel” is a semi-rigid slab or cylinder of organic polymers used as a medium for the separation of macromolecules. The gel is a substantially dilute crosslinking system and does not show flow at steady-state. Gels are, in principle, liquid by weight, but retain some properties of the liquid, such as deformability, while behaving partly as a solid due to the three-dimensional crosslinked network in the liquid. Gels are crosslinking in a fluid that gives the gel its structure (hardness) and contributes to the adhesive stick (tackiness). As a result, the gel can be regarded as a dispersion of liquid molecules in a solid, ie liquid particles dispersed in a solid medium. "Gelization time" is the time it takes for the polymerizable solution to form a gel.

A "hydrogel" is a network of polymer chains that are hydrophilic and are sometimes found as colloidal gels in which water is the dispersion medium. Hydrogels are highly absorbent (for example, polymeric networks that can contain more than 90% water) and have a very similar flexibility to natural tissues due to their significant moisture content.

“Polymer” is a macromolecule made up of a series of repeating subunits. The basic repeating subunits are known as “monomers”. As a group, polymers are known for their tensile strength and elasticity.

“Photoinitiators” are molecules that generate reactive species (free radicals, cations or anions) upon exposure to radiation (UV or visible light). The photoinitiator of the present invention induces polymerization of the polymerizable solution. Examples of photoinitiators useful in this method are lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl] -2-methylpropan-1-one (IRGACURE® 2959), eosin Y+triethanolamine, or riboflavin.

Methacrylate is an ester or salt derived from methacrylic acid. Methacrylate is a common monomer in polymer plastics, forming acrylate polymers. The addition of a methacrylate group to collagen results in a photocurable collagen methacrylate (rh collagen-MA or MA-rh collagen). The addition of a methacrylate group to hyaluronic acid (HA) results in a photocurable hyaluronic acid-methacrylate (HAMA or MA-HA).

In some embodiments, the rh collagenase used in the dermal fillers described herein comprises a combination of a non-modified rh collagen and MA-rh collagen. In some embodiments, the ratio of non-modified rh collagen to MA-rh collagen is about 1:0, 1:1, 1:2, 1:3, 1:4, 0:1, 2:1, 3: 1, or 4:1. In some embodiments, the final concentration range of MA-rh collagen comprises about 0 mg/ml to 12 mg/ml. In some embodiments, the final concentration range of non-modified rhcollagen comprises about 0 mg/ml to 12 mg/ml. In some embodiments, the final concentration range of MA-rh collagen comprises about 0 mg/ml to 12 mg/ml, and the final concentration range of non-modified rh collagen is about 0 mg/ml to 12 mg/ml. Include. In some embodiments, the final concentration range of MA-rh collagen comprises about 0 mg/ml to 6 mg/ml. In some embodiments, the final concentration range of non-modified rhcollagen comprises about 0 mg/ml to 6 mg/ml. In some embodiments, the final concentration of MA-rh collagen comprises about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg/ml. In some embodiments, the final concentration of non-modified-rh collagen comprises about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg/ml.

Thiols are organosulfur compounds containing a carbon-bonded sulfhydryl (R-SH) group, wherein R represents an alkyl or other organic substituent. Thiolation of collagen can improve adhesion and mucosal adhesion properties, and affects swelling ability.

Light is in the form of electromagnetic radiation. "Visible light" is light having a wavelength in the range of 380 nm to 800 nm or at least in the range of 390 nm to 700 nm. "Ultraviolet light" has a shorter wavelength, while "infrared" has a longer wavelength.

The lighting means may be a light source suitable for activating the photoinitiator used, and may activate the photoinitiator from outside the body. Since a thermal initiator can be used, an infrared light source can be used, and an ultraviolet-activated initiator can be used, so a suitable ultraviolet light source can be used, while a preferred light source is a white light source. Therefore, a suitable photoinitiator is used, so that the maximum absorption of the initiator and light source is tuned. As mentioned above herein, one such source of visible light is a light emitting diode (LED). Other suitable light sources can be used by applying electromagnetic radiation to the body, if necessary, to the body, at such sites as below the skin surface, or from above the skin surface, as long as gelation occurs in the body. Electromagnetic radiation is applied over time and over a period of time, at an intensity that makes gelation possible. The light source may be placed on the skin surface or directly on the skin surface, typically over the site of a shaped or shaped polymeric solution.

The monomer solutions of some embodiments will contain any of a variety of other substances as known in the pharmaceutical art, such as inert substances such as preservatives, fillers, excipients or diluents, pharmacologically active molecules or agents such as small molecules or biological agents, cells, etc. I can. Therefore, suitable inert agents or biologically active agents can be added to the monomer solution. In the case of a biologically active agent, the active agent may exert a pharmacological action locally at or near the polymerized or networked structural site of interest, or may be released from the formed scaffold, matrix or network and migrate through adjacent tissue spaces, or It can enter the circulatory system for less local effect.

As discussed above, the polymeric solution method of interest can also be used in combination with other dermatology, orthopedics, cosmetology and other medical treatments.

In some embodiments, the polymerizable solution is mixed with known fillers to provide a composition that is moldable, contourable, has a long residence time, and the like. Examples of fillers include, but are not limited to, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or modified derivatives thereof, or combinations thereof. no. In some embodiments, the polymerizable solution in the semi-liquid phase will be injected into the dermis independently as well as known fillers in the semi-liquid phase and will provide a composition that is moldable together and has a long residence time and the like. Examples of independently injectable fillers include hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or modified derivatives thereof, or combinations thereof. However, it is not limited to these. In some embodiments, the polymerizable solution in the semi-liquid phase will be injected into the dermis as a mixture, moldable together, contourable, and will provide a composition having a long residence time and the like. Examples of fillers that can be injected with rh collagen are hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or modified derivatives thereof, or combinations thereof. Including, but not limited to.

In yet another aspect, disclosed herein is a method of inducing a cellular growth promoting scaffold in a tissue space below the epidermis, the method comprising introducing a solution into the tissue space, the solution comprising (a) Plant-derived human collagen; And (b) at least one growth factor or source thereof.

In one embodiment, the source of the at least one growth factor comprises plasma or serum-rich plasma.

In one embodiment, the cellular growth promoting scaffold promotes healing or replacement due to degradation or injury of collagen-containing tissue. In one embodiment, the collagen-comprising tissue is selected from the group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones or teeth. In certain embodiments, the cellular growth promoting scaffold promotes healing of tendinitis.

In one embodiment, the plant-derived collagen comprises rh collagen. In one embodiment, the plant-derived collagen is obtained from a genetically modified plant. In various embodiments, the genetically modified plant is a genetically modified plant selected from tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. In one embodiment, the genetically modified plant is a tobacco plant.

In one embodiment, the genetically modified plant comprises an expression sequence of at least one gene sequence of human deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-alpha, P4H-beta, and LH3. Include.

In certain embodiments, the plant-derived human collagen comprises at least one modified human collagen alpha-1 chain as set forth in SEQ ID NO: 3 and expressed in a genetically modified plant; And at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO: 6 and expressed in a genetically modified plant; The genetically modified plants further express exogenous prolyl-4-hydroxylase (P4H).

In another specific embodiment, the method further comprises expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C.

In one specific embodiment, the human collagen alpha-1 chain is encoded by a sequence as set forth in SEQ ID NO: 1. In another specific embodiment, the human collagen alpha-2 chain is encoded by a sequence as set forth in SEQ ID NO: 2.

In one embodiment, the exogenous P4H is mammalian P4H. In one specific embodiment, the exogenous P4H is human P4H.

In one embodiment, the method further comprises targeting human collagen alpha-1 to the vacuoles of the plant or genetically modified plant, and degrading it to ficin. In one embodiment, the method further comprises targeting human collagen alpha-2 to the vacuoles of the plant or genetically modified plant, and degrading it to ficin.

In one specific embodiment, the plant-derived human collagen is atelocollagen.

Those of skill in the art will appreciate that the term “dermal filler” encompasses solutions comprising plant-derived human collagen, eg, type 1 recombinant human collagen (rh collagen) or derivatives thereof, in some embodiments. The term “dermal filler” is also in some embodiments, plant-derived human collagen, eg, type 1 recombinant human collagen (rh collagen) or derivatives thereof, and fillers or derivatives thereof, all of the same meaning and quality, or Comprises a solution comprising a crosslinked filler or a derivative thereof, wherein the dermal filler can be used to strengthen tissue structure, or to reduce wrinkles, folds, fine lines, wrinkles, or scars, or any combination thereof. Can be used.

Those skilled in the art will appreciate that the dermal fillers described herein are different formulations, such as but not limited to,

ㆍ rh collagen or its MA or thiol derivative;

ㆍ IPN or semi-IPN or double cross-linked networks comprising rh collagen or rh collagen-MA or rh collagen-thiol, and fillers or derivatives thereof;

ㆍ IPN or semi-IPN or double crosslinked networks comprising rh collagen or rh collagen-MA or rh collagen-thiol, and HA or MA-HA or thiol-HA;

ㆍ IPN or semi-IPN or double cross-linked networks comprising rh collagen or rh collagen-MA or rh collagen-thiol, and PVA or MA-PVA or thiol-PVA;

ㆍ IPN or semi-IPN or double-crosslinked networks comprising rhcollagen or rhcollagen-MA or rhcollagen-thiol, and PEG or MA-PEG or thiol-PEG;

ㆍ IPN or semi-IPN or double bridged networks comprising rh collagen or rh collagen-MA or rh collagen-thiol, and OC or MA-OC or thiol-OC;

ㆍ an IPN or semi-IPN or double-crosslinked network or cellular growth promoting scaffold comprising rh collagen, and an autologous platelet rich plasma (PRP) fraction of blood containing a high concentration of platelets;

ㆍ IPN or semi-IPN or double-crosslinked networks or cellular growth promoting scaffolds, each comprising an autologous platelet rich plasma (PRP) fraction of blood containing rh collagen and a high concentration of platelets, wherein the platelets are vascular- Endothelial growth factor (VEGF), transforming beta growth factor (TGF-beta), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), or IPN or semi-IPN or double cross-linked networks or cellular growth promoting scaffolds that release various types of growth factors (GF), including hepatocyte growth factor (HGF), or combinations thereof;

ㆍ A double crosslinked dermal filler comprising rh collagen or rh collagen-MA or rh collagen-thiol crosslinked to a crosslinked filler or a derivative thereof;

ㆍ Crosslinked HA or crosslinked MA-HA or crosslinked thiol-HA with crosslinked rh collagen or a double crosslinked dermal filler comprising rhcollagen-MA or rhcollagen-thiol;

ㆍ A double crosslinked dermal filler comprising crosslinked PVA or crosslinked MA-PVA or crosslinked thiol-PVA and crosslinked rh collagen or rh collagen-MA or rh collagen-thiol;

ㆍ A double-crosslinked dermal filler comprising crosslinked PEG or crosslinked MA-PEG or crosslinked thiol-PEG and crosslinked rh collagen or rhcollagen-MA or rhcollagen-thiol; or

ㆍ It will be appreciated that cross-linked OC or cross-linked MA-OC or cross-linked thiol-OC include double-crosslinked dermal fillers comprising rhcollagen or rhcollagen-MA or rhcollagen-thiol.

Those of skill in the art will appreciate that, in some embodiments, the term “cellular growth promoting scaffold” encompasses dermal fillers comprising collagen and an autologous platelet rich plasma (PRP) fraction of blood or a component thereof. In some embodiments, PRP does not include “cells”, but includes membranous vesicles (of cell origin) that contain growth factors and plasma components such as fibrinogen and pro-thrombin. In some embodiments, a “cellular growth promoting scaffold” encompasses a dermal filler comprising collagen and an autologous platelet rich plasma (PRP) fraction of blood or a component thereof, and at least an additional filler component.

In some embodiments, the cellular growth promoting scaffold may be an IPN network, a semi-IPN network, or a double-crosslinked dermal filler further comprising an autologous platelet rich plasma (PRP) fraction of blood containing a high concentration of platelets. A dermal filler, wherein the autologous PRP fraction of blood contains a high concentration of platelets, the platelets being vascular-endothelial growth factor (VEGF), transforming beta growth factor (TGF-beta), platelet-derived growth factor (PDGF). ), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF) or hepatocyte growth factor (HGF), or various types of growth factors (GF) including a combination thereof do. In some embodiments, the cellular growth promoting scaffold is, vascular-endothelial growth factor (VEGF), transforming beta growth factor (TGF-beta), platelet derived growth factor (PDGF), platelet derived epidermal growth factor (PDEGF), IPN network, semi-IPN network, or double cross-linking further comprising at least one growth factor comprising fibroblast growth factor (bFGF), epidermal growth factor (EGF) or hepatocyte growth factor (HGF), or a combination thereof Dermal fillers, including dermal fillers. In some embodiments, the cellular growth promoting scaffold comprises an IPN network further comprising a subset or fraction of PRP components, a semi-IPN network, or a dermal filler comprising a double crosslinked dermal filler.

In some embodiments, the dermal filler described herein comprises a polymerizable solution. In some embodiments, the dermal filler described herein comprises a non-polymerizable solution. In some embodiments, polymerization of the dermal filler solution occurs in vivo . In some embodiments, the components of the polymeric dermal filler solution are injected together and then polymerized to form a cured dermal filler. In some embodiments, the components of the polymerizable dermal filler solution are injected independently and then polymerized to form a cured dermal filler. One example of a unique approach of independently injectable dermal filler components is, in some embodiments, injecting a filler, such as, but not limited to, HA or a derivative thereof into the dermal dermis, and a methacrylated rh collagen or thiol-rh And separately injecting collagen into the skin dermis in the proximal vicinity of the first injection, wherein the component is in a semi-liquid phase and then crosslinked in situ . This approach, in some embodiments, allows easier injection and in-situ shaping prior to curing the dermal filler components together by photopolymerization.

In some embodiments, dermal fillers provided herein are used in a soft tissue strengthening method. In some embodiments, dermal fillers provided herein enhance cell proliferation. In some embodiments, the dermal filler provided and used in the soft tissue strengthening method degrades over time. In some embodiments, the dermal fillers provided herein are used in a method of strengthening soft tissue, wherein the dermal filler fills the tissue space below the epidermis. In some embodiments, the dermal fillers provided herein are used in a method of strengthening soft tissue, wherein the use reduces wrinkles, folds, fine lines, wrinkles, or scars.

In one embodiment, a solution comprising plant-derived human collagen has a reduced viscosity at room temperature compared to a similar solution comprising tissue-extracted human or animal-derived collagen in the same concentration and formulation. In another embodiment, a solution comprising plant-derived human collagen has a reduced viscosity at 37° C. compared to a similar solution comprising tissue-extracted human or animal-derived collagen in the same concentration and formulation. In yet another embodiment, a solution comprising plant-derived human collagen is introduced into the tissue space with reduced force at room temperature compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. Is introduced. In yet another embodiment, the solution comprising plant-derived human collagen is compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. Is introduced into In one specific embodiment, a solution comprising plant-derived human collagen has increased scaffolding formation or cell growth compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. Promotes an increase in

In yet another embodiment, disclosed herein is the use of a solution injected into the tissue space below the epidermis to induce a cellular growth promoting scaffold, wherein the solution is healing due to degradation or injury of collagen-comprising tissue or Plant-derived human collagen and at least one growth factor or source thereof to facilitate replacement. In certain embodiments, the source of at least one growth factor comprises plasma or serum-rich plasma.

In an embodiment, the collagen-comprising tissue is selected from the group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones or teeth. In another embodiment, the cellular growth promoting scaffold promotes healing of tendinitis. In an embodiment, the collagen-comprising tissue is skin.

In some embodiments, a genetically modified plant is provided, wherein the plant is capable of expressing at least one type of collagen alpha chain and accumulating the chain in subcellular compartments lacking endogenous P4H activity.

As used herein, the phrase “genetically modified plant” refers to any lower (eg, moss) or higher (related) plant or their (eg, moss) that is stably or transiently transformed with an exogenous polynucleotide sequence. For example, it refers to tissues or isolated cells) of a cell suspension. Examples of plants include tobacco, corn, alfalfa, rice, potatoes, soybeans, tomatoes, wheat, barley, canola, cotton, carrots, as well as lower plants such as moss.

As used herein, the phrase “collagen chain” refers to a collagen subunit, such as an alpha 1 or alpha 2 chain of collagen fibers, preferably type I fibers. As used herein, the phrase “collagen” refers to an assembled collagen trimer comprising two alpha 1 chains and one alpha 2 chain for type I collagen. Collagen fibers are collagen without terminal propeptides C and N.

As used herein, the phrase “subcellular compartment without endogenous P4H activity” refers to any compartmentalized region of a cell that does not contain plant P4H or an enzyme with plant-like P4H activity. Examples of such subcellular compartments include vacuoles, apoplasts and cytoplasm, as well as organelles such as chloroplasts, mitochondria, and the like.

Any type of collagen chain can be expressed by the genetically modified plants of the present invention. Examples include fibril-forming collagen (type I, II, III, V and XI), network-forming collagen (IV, VIII and X), and collagen associated with the fibrillar surface (type IX, XII). Type and type XIV), collagen (type XIII and type XVII) occurring as a transmembrane protein, or form 11-nm periodic beaded filaments (type VI).

In one embodiment, the expressed collagen chain is alpha 1 and/or alpha 2 of type I collagen. The expressed collagen alpha chain can be encoded by any polynucleotide sequence derived from any mammal. In certain embodiments, the sequence encoding the collagen alpha chain is human and is set forth in SEQ ID NO: 1 and 4 .

Typically, the alpha collagen chain expressed in plants may or may not include its terminal propeptides (ie, propeptide C and propeptide N).

Processing of procollagen by plant proteolytic activity is different from normal processing in humans, and propeptide C is removed by plant proteolytic activity, but the cleavage site is not known. Cleavage of the C propeptide can occur on the procollagen peptide prior to assembly of the trimer (binding of the three C-propeptides is essential to initiate assembly of the trimer).

N-propeptide cleavage by plant proteolytic activity occurs in mature plants, but not in seedlings. This cleavage removes 2 amino acids (2 of 17) from the N telopeptides.

C-propeptides (and to a lesser extent N-propeptides) keep these procollagens soluble during passage of procollagens through animal cells (Bulleid et al., 2000) and are expected to have similar effects in plant cells. . After or during the secretion of the procollagen molecule into the extracellular matrix, the propeptide is removed by the procollagen N-proteinase and C-proteinase, thereby allowing the spontaneous self-assembly of the collagen molecule into fibrils. Triggers. Removal of the propeptide by procollagen N-proteinase and C-proteinase is necessary and sufficient to lower the solubility of procollagen by more than 10,000-fold and to initiate self-assembly of collagen into fibers. . Decisive in this assembly process is a short non-triple-helical peptide called telopeptide at the end of the triple-helix domain, which ensures the correct covalent registration of collagen molecules within the fibrillar structure and allows their crosslinkable aldehydes. It acts to lower the critical concentration of self-assembly. Pepsin can cleave propeptides during collagen production. However, pepsin damages telopeptides, and as a result, pepsin-extracted collagen cannot form an ordered fibrillar structure.

Protein disulfide isomerase (PDI), which forms the beta subunit of human P4H, has been shown to bind to C-propeptide prior to trimer assembly, thereby also acting as a molecular chaperone during chain assembly.

The use of human procollagen I N-proteinase and procollagen C-proteinase expressed in different plants will produce collagen that is more similar to native human collagen and capable of forming ordered fibrillar structures. I can.

When N peptide or C propeptide or both are included in the expressed collagen chain, the genetically modified plants of the invention can also express the respective protease (ie, C or N or both). Polynucleotide sequences encoding these proteases are exemplified by SEQ ID NO: 18 (protease C) and 20 (protease N). These proteases can be expressed so that these proteases accumulate in the same subcellular compartment as the collagen chain.

Accumulation of expressed collagen chains in subcellular compartments without endogenous P4H activity can be accomplished through any one of several approaches.

For example, the expressed collagen chain may comprise a signal sequence for targeting the expressed protein to a subcellular compartment such as an apoplast or organelle (eg, chloroplast). Examples of suitable signal sequences include chloroplast transport peptides (included in Swiss-Prot entry P07689, amino acids 1 to 57) and mitochondrial transport peptides (included in Swiss-Prot entry P46643, amino acids 1-28). The Examples section that follows provides additional examples of suitable signal sequences, as well as guidelines for using these signal sequences in the expression of collagen chains in plant cells.

Alternatively, the sequence of collagen chains can be modified in such a way as to alter the cellular localization of collagen when expressed in plants.

As mentioned above, plant ER contains P4H, which cannot properly hydroxylate collagen chains. The collagen alpha chain naturally contains an ER targeting sequence that directs this expressed collagen into the ER into which the collagen is modified post-translational (including false hydroxylation). Therefore, removal of the ER targeting sequence will cause the collagen chains to accumulate in the cytoplasm, and these collagen chains are free of any post-translational modifications including any hydroxylation.

Example 1 in the Examples section that follows describes the generation of a collagen sequence without an ER sequence.

More alternatively, collagen chains can be expressed and accumulated in DNA containing organelles such as chloroplasts or mitochondria. Further explanation of chloroplast expression is provided herein below.

As mentioned above, hydroxylation of the alpha chain is required for the assembly of stable type I collagen. Since the alpha chains expressed by the genetically modified plants of the present invention accumulate in compartments without endogenous P4H activity, these chains must be isolated from plants, plant tissues or cells and hydroxylated in vitro. This hydroxylation can be achieved by the method described by Turpeenniemi-Hujanen and Myllyla (Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro. Biochim Biophys Acta. 1984 Jul. 16; 800 (1). ):59-65]).

While this in vitro hydroxylation can lead to correctly hydroxylated collagen chains, this can be difficult and expensive to achieve.

In order to overcome the limitations of in vitro hydroxylation, the genetically modified plants of the invention preferably also correctly hydroxylate the collagen alpha chain(s) (i.e., the proline (Y) position of the Gly-XY triplet). It co-expresses P4H, which is capable of hydroxylation only. P4H is an enzyme consisting of two subunits, alpha and beta. While both are required to form an active enzyme, the beta subunit also possesses chaperone function.

The P4H expressed by the genetically modified plants of the present invention is preferably human P4H encoded by eg SEQ ID NO:12 and 14 . In addition, P4H mutants, or P4H homologues, exhibiting enhanced substrate specificity can also be used.

Suitable P4H homologues are exemplified by Arabidopsis oxidoreductase, identified by NCBI deposit NP_179363. The pairwise alignment of this protein sequence and the human P4H alpha subunit, performed by the inventors, revealed the highest homology between the functional domains of any known P4H homolog in plants.

Since P4H needs to be co-accumulated with the expressed collagen chain, its coding sequence is preferably modified accordingly (addition of signal sequences, deletions that may prevent ER targeting, etc.).

In mammalian cells, collagen is also modified by lysyl hydroxylase, galactosyltransferase and glucosyltransferase. These enzymes sequentially transform lysyl residues into hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. A single human enzyme, lysyl hydroxylase 3 (LH3), is capable of catalyzing all three successive steps in hydroxylysine linked carbohydrate formation.

Therefore, the genetically modified plants of the present invention preferably also express mammalian LH3. As set forth by SEQ ID NO: 22 The LH3 coding sequence can be used for this purpose.

The collagen chain(s) and modifying enzymes described above are stably integrated comprising polynucleotide sequences encoding alpha chains and/or modifying enzymes (e.g., P4H and LH3) placed under the transcriptional control of plant functional promoters. It can be expressed from a nucleic acid construct or a transiently expressed nucleic acid construct. Such nucleic acid constructs (also referred to herein as expression constructs) may be placed for expression throughout a whole plant, a defined plant tissue or a defined plant cell, or at a defined stage of development of the plant. Such constructs may also include selectable markers (eg, antibiotic resistance), enhancer elements, and origins of replication for bacterial replication.

Constructs comprising two expressible inserts (e.g., two types of alpha chains, or an alpha chain and P4H) preferably comprise a separate promoter for each insert, or alternatively such constructs are single It will be appreciated that it is possible to express a single transcript chimera comprising both insertion sequences from the promoter. In this case, the chimeric transcript includes an IRES sequence between the two insert sequences, allowing downstream inserts to be translated therefrom.

Many plant functional expression promoters and enhancers, which may be tissue specific, developmentally specific, constitutive or inducible, can be used by the constructs of the invention, some examples provided herein below.

As used herein, in the following specification and claims sections, the phrase “plant promoter” or “promoter” includes a promoter capable of directing gene expression in plant cells (including DNA containing organelles). Such promoters can be derived from plant, bacterial, viral, fungal or animal origin. Such promoters can be constitutive, i.e., can direct high levels of gene expression in a plurality of plant tissues, or such promoters can be tissue specific, i.e., can direct gene expression in specific plant tissues or tissues. Alternatively, such promoters may be inducible, ie, may direct gene expression under stimulation, or such promoters may be chimeric, ie, may be formed as part of at least two different promoters.

Therefore, the plant promoter used may be a constitutive promoter, a tissue specific promoter, an inducible promoter, or a chimeric promoter.

Examples of constitutive plant promoters include, but are not limited to, CaMV35S and CaMV19S promoters, FMV34S promoters, sugarcane bacilliform badnavirus promoters, CsVMV promoters, Arabidopsis ACT2/ACT8 actin promoters, Arabidopsis ubiquitin UBQI promoters, barley leaves Thionine BTH6 promoter, and rice actin promoter.

Examples of tissue-specific promoters include, but are not limited to, soy phaseolin storage protein promoter, DLEC promoter, PHS promoter, zein storage protein promoter, conglutin gamma promoter from soybean, AT2S1 gene promoter. , The ACT11 actin promoter from Arabidopsis, the napA promoter from Brassica napus, and the potato patatin gene promoter.

Inducible promoters are promoters that are induced by specific stimuli or in the case of pathogenicity, such as stress conditions including, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions, and non- Limited, a light-inducible promoter derived from the pea rbcS gene, a promoter from the alfalfa rbcS gene, the drought-active promoters DRE, MYC and MYB; Promoters INT, INPS, prxEa, Ha hsp17.7G4 and RD21, which are active in high salinity and osmotic shock, and the promoters hsr203J and str246C, which are active in pathogenic stress.

Preferably, the promoter used by the present invention is a strong constitutive promoter that allows overexpression of the construct insert to be carried out after plant transformation.

It will be appreciated that any construct type used in the present invention can be co-transformed into the same plant using the same or different selectable markers in each construct type. Alternatively, the first construct type can be introduced into the first plant, while the second construct type can be introduced into a second isogenic plant, and the transgenic plant resulting therefrom can be They can be crossed and offspring can be selected for double transformants. Additional self-crossing of these offspring can be used to create lines that are homozygous for both constructs.

Various methods exist for introducing nucleic acid constructs into both monocotyledonous and dicotyledenous plants (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (199191) ) 42:205-225]; [Shimamoto et al., Nature (1989) 338:274-276]). These methods rely on the stable integration of the nucleic acid construct or portions thereof into the plant genome, or transient expression of the nucleic acid constructs whose sequences are not inherited by the progeny of the plant.

In addition, there are several ways in which nucleic acid constructs can be introduced directly into DNA-containing organelles such as DNA or chloroplasts.

There are two principle methods of carrying out the stable genomic integration of exogenous sequences, such as inclusion in the nucleic acid constructs of the invention into the plant genome:

(i) Agrobacterium -mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486]; [Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) p. 2-25]; [Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, CJ, Butterworth Publishers, Boston, Mass. (1989) p. 93-112].

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68]; Methods of direct absorption of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNA uptake induced by brief electric shock of Plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into Plant cells or Tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563]; [McCabe et al. Bio/Technology (1988) 6:923-926]; [Sanford, Physiol. Plant. (1990) 79:206-209]; By use of a micropipette system: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36]; [Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217]; Alternatively, by direct incubation of DNA with germinated spores, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209]; And [Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719].

The Agrobacterium system involves the use of plasmid vectors containing defined DNA sequences that are integrated into plant genomic DNA. The method of inoculation of plant tissues depends on the plant species and Agrobacterium delivery system. A widely used approach is a leaf disc procedure that can be performed with any tissue explant that provides a good source for the initiation of whole plant differentiation. Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9]. An auxiliary approach uses the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is particularly viable in the production of transgenic dicot plants.

There are various methods of direct DNA into plant cells. In electrophoresis, the protoplast is briefly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into cells using a very small micropipette. In microparticle collisions, DNA is adsorbed onto microprojectiles such as magnesium sulfate crystals, tungsten particles or gold particles, which are physically accelerated into cells or plant tissues.

After transformation, plant propagation is carried out. The most common method of plant reproduction is by seed. However, regeneration by seed propagation has the defect that crops lack uniformity due to heterozygosity, as seeds are produced by plants according to genetic variance governed by Mendelian law. Basically, each seed is genetically different, and each will grow with its own specific properties. Therefore, it is preferred that the plant to be regenerated is a transformed plant to have the same properties and characteristics of the parent transgenic plant. Thus, it is preferred that the transformed plant is regenerated by micropropagation, which provides rapid and consistent reproduction of the transformed plant.

Transient expression methods that can be used to temporarily express the isolated nucleic acid contained in the nucleic acid construct of the present invention are as described above, but microinjection and collision under conditions favoring transient expression, and packaging comprising the nucleic acid construct Viral-mediated expression in which a recombinant or unpackaged viral vector is used to infect plant tissues or cells so that the reproductive recombinant virus established therein expresses a non-viral nucleic acid sequence. .

Viruses that have been shown to be useful for transformation of plant hosts include CaMV, TMV and BV. Transformation of plants using plant viruses was conducted in US Patent No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 ( BV); And Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudoviral particles for use in expressing foreign DNA in many hosts, including plants, are described in WO 87/06261.

Construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences in plants is described in this reference as well as in Dawson, W. O. et al., Virology (1989) 172:285-292; [Takamatsu et al. EMBO J. (1987) 6:307-311]; [French et al. Science (1986) 231:1294-1297]; And [Takamatsu et al. FEBS Letters (1990) 269:73-76].

If the virus is a DNA virus, construction can be done on the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of constructing the desired viral vector with foreign DNA. Thereafter, the virus can be excised from the plasmid. If the virus is a DNA virus, the origin of replication of the bacteria can be attached to the viral DNA, which is then replicated by the bacteria. The transcription and translation of this DNA will produce a coat protein, which will encapsidate the viral DNA. When the virus is an RNA virus, the virus is usually cloned as cDNA and inserted into a plasmid. After that, the plasmid is used to make all constructs. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translating the viral gene to produce the envelope protein(s) that encapsulate the viral RNA.

Construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences as included in the constructs of the present invention in plants is demonstrated by this reference as well as US Pat. No. 5,316,931.

In one embodiment, the native capsular protein coding sequence has been deleted from the viral nucleic acid, can be expressed in a plant host, package the recombinant plant viral nucleic acid, and ensure systemic infection of the host by the recombinant plant viral nucleic acid non-native plant A plant viral nucleic acid is provided in which the viral coat protein coding sequence and a non-native promoter, preferably a subgenomic promoter of the non-native coat protein coding sequence, have been inserted. Alternatively, the envelope protein gene can be inactivated by insertion of a non-native nucleic acid sequence therein, so that the protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing a contiguous gene or nucleic acid sequence in a plant host and is not capable of recombining with each other and with a native subgenomic promoter. Non-native (foreign) nucleic acid sequences can be inserted adjacent to the native plant viral subgenomic promoter or native and non-native plant viral subgenomic promoters if more than one nucleic acid sequence is included. Non-native nucleic acid sequences are transcribed or expressed in a host plant under the control of a subgenomic promoter to produce the desired product.

In a second embodiment, the recombinant plant viral nucleic acid comprises a first, except that the native coat protein coding sequence is disposed adjacent to one of the non-native coat protein subgenomic promoters instead of the non-native coat protein coding sequence. It is provided as in the embodiment.

In a third embodiment, a recombinant plant viral nucleic acid is provided in which a native coat protein gene is adjacent to its subgenomic promoter and at least one non-native subgenomic promoter has been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in the plant host and are not capable of recombining with each other and with native subgenomic promoters. The non-native nucleic acid sequence can be inserted adjacent to the non-native subgenomic plant virus promoter such that the sequence is transcribed or expressed in a host plant under the control of the subgenomic promoter to produce the desired product.

In a fourth embodiment, the recombinant plant viral nucleic acid is provided as in the third embodiment, except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

The viral vector is encapsulated by the envelope protein encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. Recombinant plant virus nucleic acids or recombinant plant viruses are used to infect suitable host plants. Recombinant plant viral nucleic acids can be replicated in a host, spread systemically in the host, and transcribed or expressed the foreign gene(s) (isolated nucleic acid) in the host to produce the desired protein.

Techniques for introducing exogenous nucleic acid sequences into the chloroplast genome are known. This technique involves the following procedure. First, plant cells are chemically treated, reducing the number of chloroplasts per cell by about 1. Thereafter, the exogenous nucleic acid is introduced into the particle via particle collision for the purpose of introducing at least one exogenous nucleic acid molecule into the chloroplast. The exogenous nucleic acid is chosen to be capable of integrating into the genome of the chloroplast through homologous recombination, which is easily carried out by enzymes unique to the chloroplast. To this end, the exogenous nucleic acid includes, in addition to the gene of interest, at least one nucleic acid stretch derived from the chloroplast genome. In addition, the exogenous nucleic acid comprises a selection marker, which after such selection serves by a sequential selection procedure to ascertain whether all or substantially all copies of the chloroplast will contain the exogenous nucleic acid. Further details regarding this technique are found in U.S. Patent Nos. 4,945,050 and 5,693,507, which are incorporated herein by reference. Therefore, the polypeptide can be produced by the protein expression system of the chloroplast and integrated into the inner membrane of the chloroplast.

The transformation approach described above can be used to produce collagen chains and/or modifying enzymes, as well as assembled collagen (with or without propeptide), in any plant species, or plant tissues derived therefrom, or isolated plant cells. have.

Preferred plants are those capable of accumulating large amounts of the collagen chains, collagen and/or processing enzymes described herein. Such plants may also be selected according to their tolerance to stress conditions and the ease with which the expressed components or assembled collagen can be extracted. Examples of preferred plants include tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola and cotton.

Collagen fibers are widely used in the food and beauty industries. Therefore, although collagen fiber components (alpha chains) and modifying enzymes expressed by plants are used for the industrial synthesis of collagen, complete collagen production in plants is preferred in terms of its simplicity and cost effectiveness.

Several approaches can be used to produce type I collagen in plants. For example, collagen alpha 1 chain is isolated from plants expressing collagen alpha 1 and P4H (and optionally LH3), and plants expressing collagen alpha 2 and P4H (and optionally LH3 and protease C and/or N). Can be mixed with collagen alpha 2 chains isolated from. Because the collagen alpha 1 chain itself assembles into triple helices, it may be necessary to denature these homo-trimers before mixing and restoring with the collagen alpha 2 chain.

Preferably, the first plant expressing collagen alpha 1 and P4H (and optionally LH3 and protease C and/or N) is to be crossed with a second (and preferably allogeneic) plant expressing collagen alpha 2. Alternatively, a first plant expressing both alpha chains can be crossed with a second plant expressing P4H and optionally LH3 and protease C and/or N.

While the plant breeding approach described above uses two individually transformed plants, it is noted that approaches using three or more individually transformed plants, each expressing one or two components, can also be used. It should be noted.

One of skill in the art would be familiar with various plant breeding techniques, and such further descriptions of such techniques are not provided herein.

Although the plant breeding approach is preferred, a single plant expressing collagen alpha 1 and 2, P4H and LH3 (and optionally protease C and/or N) is designed to introduce one or more expressible components into cells, each Can be generated through several transformation events. In this case, the stability of each transformation event can be confirmed using specific selection markers.

In any case, transformation and plant breeding approaches can be used to generate any plant that expresses any number of components. Currently preferred are plants expressing collagen alpha 1 and 2 chains, P4H, LH3 and at least one protease (eg, protease C and/or N). As further described in the Examples section that follows, these plants accumulate collagen, which exhibits stability at temperatures up to 42°C.

Progeny from breeding or alternatively multi-transformed plants can be selected by confirming the presence of exogenous mRNA and/or polypeptides using nucleic acid or protein probes (eg, antibodies). The latter approach is preferred because it allows localization of the expressed polypeptide components (eg, by probing fractionated plant extracts), thus also confirming the potential for correct processing and assembly. Examples of suitable probes are provided in the Examples section that follows.

Once the collagen-expressing progeny are identified, these plants are further cultured under conditions that maximize the expression of collagen chains as well as modifying enzymes.

Since such free proline accumulation can facilitate the production of different proline-rich proteins comprising collagen chains expressed by the genetically modified plants of the present invention, preferred culture conditions increase free proline accumulation in cultured plants. It is a condition to let.

Accumulates in a variety of plants in response to a wide range of environmental stresses including water depletion, salinization, low and high temperatures, pathogen infections, heavy metal toxicity, anaerobiosis, nutrient deficiencies, air pollution and UV-irradiation (Hare). And Cress, 1997).

Free proline may also accumulate in response to treatment of plants or soil with compounds such as ABA or stress inducing compounds such as copper salts, paraquate, salicylic acid, and the like.

Therefore, collagen-expressing progeny can be grown under different stress conditions (eg, different concentrations of NaCl in the range of 50 mM to 250 mM or less). To further enhance collagen production, the effects of various stress conditions on collagen expression will be examined and optimized for plant viability, biomass and collagen accumulation.

Plant tissue/cells are preferably harvested upon maturation and collagen fibers are isolated using well-known prior art extraction approaches, one such approach is detailed below.

The leaves of the transgenic plant are ground to powder under liquid nitrogen, and the homogenate is extracted for 60 hours at 4° C. in 0.5 M acetic acid containing 0.2 M NaCl. Insoluble matter is removed by centrifugation. The supernatant containing the recombinant collagen is salt-fractionated in 0.4 M and 0.7 M NaCl. The 0.7 M NaCl precipitate containing recombinant heterotrimeric collagen is dissolved in 0.1 M acetic acid, dialyzed against it, and stored at -20°C (according to Ruggiero et al., 2000).

In one embodiment, disclosed herein is a method of processing a procollagen to produce a homogeneous, soluble, fibril-forming atelocollagen.

In some embodiments, as presented herein by analysis of proteolytic results by SDS PAGE, certain plant-derived proteases (e.g., papain) are helical regions (telopeptides from telocollagen they originate from animal sources). While it is not possible to cleave the propeptide portion from the soluble procollagen without proteolytic cleavage within), other proteases (e.g., Esperase, Sabinase) effectively remove the propeptide region from the soluble procollagen. It does not cut, thereby preventing effective fibril formation. Through meticulous experimentation, the inventors have found that only certain plant-derived proteases such as ficin, and bacterial-derived proteases such as neutrase and subtilisin, are used to correctly cleave the propeptide moiety (including telopeptides) from the soluble procollagen. It has been found that it is possible to produce homogeneous preparations of soluble atelocollagens ( FIGS. 13, 15, 17, 19 and 20 ) without breaking down the helical regions of non-animal procollagens. Furthermore, the inventors have shown that recombinant trypsin can also make correct cleavage ( FIG. 26 ). The inventors further showed that cleavage by escape can cause the resulting atelocollagen to retain its fibrillar capacity (Table 5 in the Examples section below).

Therefore, according to one aspect, a method of producing atelocollagen is provided. The method comprises the step of contacting human recombinant telopeptide-comprising collagen with a protease selected from the group consisting of neutrase, subtilisin, recombinant trypsin, recombinant pepsin and ficin, wherein the human recombinant telopeptide-comprising collagen Is expressed in non-animal cells, thereby producing atelocollagen.

As used herein, the phrase “telopeptide-comprising collagen” refers to a soluble collagen molecule comprising a telopeptide longer than the telopeptide residue contained in atelocollagen. Therefore, the telopeptides-comprising collagen may be a procollagen comprising a full-length propeptide. Alternatively, the telopeptide-comprising collagen may be a procollagen molecule comprising a partially degraded propeptide. More alternatively, the telopeptide-comprising collagen may be telocollagen.

As used herein, the term “procollagen” refers to a collagen molecule (eg, human) comprising an N-terminal propeptide, a C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are set forth in SEQ ID NO: 30, 31, 36 and 37 .

As used herein, the term “telocollagen” refers to a collagen molecule that is typically included in procollagen but lacks both the N-terminal and C-terminal propeptides which still contain telopeptides. As mentioned herein in the background section above, the telopeptides of fibrillar collagen are the residues of the N-terminal propeptide and C-terminal propeptide after degradation by native N/C proteinase.

Recombinant human telocollagen can be produced in cells that have been transformed to express both the exogenous human procollagen and the respective protease (ie, C or N or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NO: 39 (protease C) and SEQ ID NO: 40 (protease N). These proteases can be expressed such that these proteases accumulate in the same subcellular compartment as the collagen chain, as further described herein below.

As used herein, the term "atelocollagen" is devoid of both the N-terminal and C-terminal propeptides and at least a portion of their telopeptides, typically included in procollagens, but under suitable conditions it is It refers to a collagen molecule that contains a sufficient portion of its telopeptides to be able to form fibrils.

Any type of atelocollagen can be produced according to the methods disclosed herein. Examples include fibril-forming collagen (type I, II, III, V and XI), network-forming collagen (IV, VIII and X), and collagen associated with the fibrillar surface (type IX, XII). Type and type XIV), collagen (type XIII and type XVII) generated as a transmembrane protein, or form 11-nm periodic bead-type filaments (type VI). According to one embodiment, the atelocollagen comprises the alpha 1 and/or alpha 2 chain of type I collagen.

In some embodiments, disclosed herein is a genetically modified form of collagen/atellocollagen--eg, collagenase-resistant collagen, and the like.

Recombinant human procollagen or telocollagen can be expressed in any non-animal cell including, but not limited to, plant cells and other eukaryotic cells such as yeast and fungi.

Plants from which human procollagen or telocollagen can be produced (i.e., can be expressed) can be of lower (e.g., moss and algae) or higher (related) plant species and tissues or isolated cells and extracts (e.g. For example, cell suspension). Preferred plants are plants that are capable of accumulating large amounts of collagen chains, collagen and/or processing enzymes described herein below. Such plants may also be selected according to their tolerance to stress conditions and the ease with which the expressed components or assembled collagen can be extracted. Examples of plants in which human procollagen can be expressed include, but are not limited to, tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, lettuce and cotton.

Recombinant human procollagen is typically performed by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.

Exemplary polynucleotide sequences encoding human procollagen are set forth in SEQ ID NOs: 32, 33, 41, and 42 .

As mentioned, the production of human telocollagen is typically carried out by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen and at least one exogenous polynucleotide sequence encoding a related protease.

The stability of the triple-helix structure of collagen requires the hydroxylation of proline by the enzyme prolyl-4-hydroxylase (P4H) to form a hydroxyproline residue within the collagen chain. Although plants are able to synthesize hydroxyproline-containing proteins, the prolyl hydroxylase responsible for the synthesis of hydroxyproline in plant cells exhibits a relatively loose substrate sequence specificity compared to mammalian P4H. Therefore, the production of collagen containing hydroxyproline only at the Y position of the Gly-X-Y triplet requires plant co-expression of collagen and human or mammalian P4H genes.

Therefore, according to one embodiment, the procollagen or telocollagen is expressed in the subcellular compartment of the plant without endogenous P4H activity. As used herein, the phrase “subcellular compartment without endogenous P4H activity” refers to any compartmentalized region of a cell that does not contain plant P4H or an enzyme with plant-like P4H activity. According to one embodiment, the subcellular compartment is a vacuole.

Accumulation of expressed procollagen in subcellular compartments without endogenous P4H activity can be affected through any one of several approaches.

For example, the expressed procollagen/telocollagen chain may comprise a signal sequence for targeting the expressed protein to a subcellular compartment such as an apoplast or organelle (eg, chloroplast). Examples of suitable signal sequences include chloroplast transport peptides (included in Swiss-Prot entry P07689, amino acids 1 to 57) and mitochondrial transport peptides (included in Swiss-Prot entry P46643, amino acids 1-28).

Alternatively, the sequence of the procollagen can be modified in such a way as to alter the cellular localization of the procollagen when expressed in plants.

In some embodiments, both human procollagen and P4H co-express and are capable of correctly hydroxylating the procollagen alpha chain(s) (i.e., only the proline (Y) position of the Gly-XY triplet). Genetically modified cells are disclosed herein. P4H is an enzyme consisting of two subunits, alpha and beta, as shown in Genbank numbers P07237 and P13674. While both subunits are required to form an active enzyme, the beta subunit also possesses chaperone function.

The P4H expressed by the genetically modified cells of the present invention is preferably human P4H encoded by, for example, SEQ ID NO:34 and 35 . In addition, P4H mutants, or P4H homologs, that exhibit enhanced substrate specificity can also be used. Suitable P4H homologs are exemplified by Arabidopsis oxidoreductase identified by NCBI deposit NP__179363.

Since it is essential that P4H is co-accumulated with the expressed procollagen chain, its coding sequence is preferably modified accordingly (eg, by addition or deletion of a signal sequence, etc.).

In mammalian cells, collagen is also modified by lysyl hydroxylase, galactosyltransferase and glucosyltransferase. These enzymes sequentially transform lysyl residues into hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues at specific positions. A single human enzyme, lysyl hydroxylase 3 (LH3), as shown in Genbank No. O60568, is capable of catalyzing all three successive steps as shown in hydroxylysine-linked carbohydrate formation.

Therefore, the genetically modified cells disclosed herein can also express mammalian LH3. As set forth by SEQ ID NO: 38 The LH3 coding sequence can be used for this purpose.

The procollagen(s) and modifying enzymes described above are stably integrated comprising a polynucleotide sequence encoding a procollagen alpha chain and/or modifying enzymes (e.g., P4H and LH3) placed under the transcriptional control of a functional promoter. Can be expressed from a modified nucleic acid construct or a transiently expressed nucleic acid construct. Such nucleic acid constructs (also referred to herein as expression constructs) can be placed for expression throughout the entire organism (e.g., plants, confined plant tissues or confined cells) and/or at defined stages of development of the organism. have. Such constructs may also include selectable markers (eg, antibiotic resistance), enhancer elements, and origins of replication for bacterial replication.

Constructs comprising two expressible inserts (e.g., two procollagen alpha chain types, or a procollagen alpha chain and P4H) preferably comprise separate promoters for each insert, or alternatively such It will be appreciated that the construct can express a single transcript chimera comprising both insertion sequences under a single promoter. In this case, the chimeric transcript may comprise an intraribosomal entry region (IRES) sequence between the two insert sequences so that downstream inserts can be translated therefrom.

Many functional expression promoters and enhancers, which may be tissue specific, developmentally specific, constitutive or inducible, can be used by the constructs of the present invention, some examples provided herein below.

Regardless of the transformation technique used, once a procollagen-expressing progeny is identified, such plants are further cultured under conditions that maximize their expression. Progeny from transformed plants can be selected by confirming the presence of exogenous mRNA and/or polypeptide using nucleic acid or protein probes (eg, antibodies). Because the latter approach allows localization of the expressed polypeptide components (e.g. by probing fractionated plant extracts), it also confirms the plant's potential for correct processing and assembly of foreign proteins. desirable.

After cultivation of such plants, telopeptides-comprising collagen is typically collected. Plant tissue/cells are preferably harvested upon maturity and procollagen fibers are isolated using an extraction approach. Preferably, the collection is carried out so that the procollagen remains in a state that can be cleaved by the protease enzyme. According to one embodiment, the crude (crude) extract is produced from the transgenic plant of the present invention, and subsequently contacted with a protease enzyme. Exemplary methods for producing crude plant extracts are described herein in the Examples section below.

It will be appreciated that the propeptide or telopeptide-comprising collagen may be purified from the genetically engineered cells of the invention prior to incubation with the protease, or alternatively may be purified after incubation with the protease. More alternatively, the propeptide or telopeptide-comprising collagen may be partially purified prior to protease treatment, and then completely purified after protease treatment. More alternatively, the propeptide or telopeptide-comprising collagen can be treated with a protease simultaneously with other extraction/purification procedures.

Exemplary methods for purifying or semi-purifying the telopeptide-containing collagen of the present invention include, but are not limited to, salting out using ammonium sulfate and/or the removal of small molecules by ultrafiltration.

As described herein in the background above, there are risks involved in the use of animal source materials for medical purposes. This risk is also relevant when selecting the proteolytic enzyme used to process the procollagen expressed in plants into atelocollagen. Application of an animal-derived source enzyme such as trypsin or pepsin can itself contaminate the final formulation with disease carriers. Therefore, it is desirable to design a production system in which all components are free of animal sources.

It has been disclosed herein that only certain proteases can correctly cleave recombinant propeptide or telopeptide-containing collagen. These include certain plant-derived proteases such as ficin (EC 3.4.22.3) and certain bacterial-derived proteases such as subtilisin (EC 3.4.21.62), neutrase. In some embodiments, disclosed herein is the use of recombinant enzymes such as rhtrypsin and rhpepsin. Such enzymes, e.g., ficin from Fig tree latex (Sigma, catalog #F4125 and Europe Biochem), subtilisin from Bacillus licheniformis (Sigma, catalog #P5459), Neutrase from Bacterium Bacillus amyloliquefaciens (Novozymes, catalog #PW201041), and TrypZean, a recombinant human trypsin expressed in maize. TM (Sigma catalog #T3449) is commercially available.

The procollagen or telocollagen is preferably contacted with the protease under conditions that allow the protease to cleave the propeptide or telopeptides therefrom. Typically, conditions depend on the specific protease selected. Therefore, for example, procollagen can be incubated with the protease for up to 15 hours at a concentration of 1 mg/ml to 25 mg/ml and a temperature of about 10° C. to 20° C.

After protease digestion, the resulting atelocollagen can be further purified by salt precipitation, e.g., as described in the Examples section below, so that the final product is neutrase, subtilisin, ficin and recombinant human trypsin. Atelocollagen processed from plant or plant-cell-generated procollagen by a protease selected from the group consisting of and analyzed using methods known in the art (e.g., size analysis via Coomassie staining, Western analysis, etc.) It includes a purified composition of.

After purification, the atelocollagen can be re-solubilized by addition of an acidic solution (eg 10 mM HCl). This acidic solution is useful for the storage of purified atelocollagen.

The present inventors have shown that after decomposition by ficin, atelocollagen maintains the ability of the atelocollagen to form fibrils upon neutralization of the acid solution described above. According to one embodiment, at least 70% of the purified and re-solubilized atelocollagen produced according to the method of the present invention may form fibrils. According to one embodiment, at least 88% of the purified and re-solubilized atelocollagen produced according to the method of the present invention may form fibrils.

The ability to form fibrils is useful for medical purposes, including, but not limited to, cosmetic surgery, aid in healing of burn patients, bone and reconstruction of a wide variety of dental, dental and surgical purposes.

As noted in the background section, type I collagen is considered a perfect candidate for use as a major component of building materials in 3D-bioprinting. Despite the significant benefits imparted by these natural polymers, many factors hinder their use for 3D bioprinting. The use of tissue-extracted collagen for this purpose is limited due to its sensitivity to temperature and ionic strength, driving spontaneous gel formation under physiological conditions at temperatures above 20°C [eg, PureCol, Advanced BioMatrix, Inc. Reference]. The typical temperature-dependent formation of the tissue extracted-collagen gel significantly impedes the correct flowability during printing. Keeping the printing medium at a low temperature until application is a possible solution to this phenomenon, but implies serious technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not become gel-like under these conditions. However, gelatin lacks the real tissue and cell interactions of native collagen, resulting in loss of significant biological functions.

Recent technological advances are the introduction of five human genes encoding heterotrimeric type I collagen into tobacco plants, thereby introducing non-contact human type I collagen (rh collagen) (COLLPLANT ^™ , Israel; also SIGMA-ALDRICH, St. Louis, Missouri, USA). (Available from ®) resulting in the development of a system for purification. Proteins are purified to homogeneity through cost-effective industrial processes that utilize the unique properties of collagen. Reference is also made to WO 2006/035442, WO 2009/053985, and patents and patent applications derived therefrom, all of which are incorporated by reference in their entirety as set forth herein.

Therefore, according to one aspect, disclosed herein is a genetically modified plant, the plant is capable of expressing at least one type of collagen alpha chain and accumulating the chain in a subcellular compartment without endogenous P4H activity. .

Type I collagen and rhcollagen are considered candidates for use as major components of building materials in 3D-bioprinting. Various types of scaffolding have been used in cosmetic and other reconstruction applications.

In addition, the use of dermal fillers for strengthening soft tissues, for example reducing wrinkles, has increased. One possible method of using dermal fillers involves injecting a polymerizable dermal filler material into a desired area, followed by contouring or shaping the filler into the desired shape. Polymerization and crosslinking of the material by one of a variety of methods can transform monomers in the injected material to form polymers and chains, which can form networks and retain the desired shaped shape. There are many ways to form polymers and crosslink polymers. Other methods involve photo-reactive reagents and photo-induced reactions that generate reactive species in monomer solutions.

However, at least some of these approaches continue to focus on tissue-derived collagen or non-collagen polymers (eg, poly(vinyl alcohol) or hyaluronic acid). Moreover, the use of tissue-extracted collagen is limited due to its sensitivity to temperature and ionic strength, which drives spontaneous gel formation under physiological conditions at temperatures above 20°C [eg, PureCol, Advanced BioMatrix, Inc. Reference]. The typical temperature-dependent formation of the tissue extracted-collagen gel significantly impedes the correct flowability. Keeping collagen at low temperatures until application is a possible solution to this phenomenon, but implies serious technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not become gel-like under these conditions. However, gelatin lacks the real tissue and cell interactions of native collagen, resulting in loss of significant biological functions. Moreover, the viscosity makes it more difficult to be injected under the dermis using micro-gauge needles, and also makes it more difficult to diffuse and mold gelatin into smaller cavities.

Embodiments of the dermal fillers and uses thereof disclosed herein,

1. A method of filling the tissue space under the epidermis, the method comprising:

a. Introducing a polymerizable solution into the tissue space, wherein the polymerizable solution,

i. Crosslinkable, plant-derived human collagen; And

ii. Comprising a photoinitiator; And

b. Inducing polymerization by applying light to the surface of the epidermis superficial to the space.

2. A method of filling the tissue space under the epidermis, the method comprising:

(a) shaping or shaping the polymerizable solution into the desired arrangement within the tissue space, wherein the step is simultaneous or subsequent to the applying light.

3. A method of filling the tissue space under the epidermis, wherein the shaping or shaping step reduces wrinkles, folds, fine lines, wrinkles or scars.

4. A method of filling the tissue space under the epidermis, wherein the crosslinkable, plant-derived human collagen is methacrylated or thiolated.

5. As a method of filling the tissue space under the epidermis, the polymer solution is hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof. Derivatives, oxidized cellulose (OC) or modified derivatives thereof, polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or A method further comprising a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

6. A method of filling the tissue space under the epidermis, wherein hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) ) Microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or modified derivatives of crystalline nanocellulose (CNC), comprising photopolymerizable modified derivatives.

7. Hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres according to item 5, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or a modified derivative of crystalline nanocellulose (CNC) comprises a methacrylated derivative or a thiolated derivative.

8. A method of filling a tissue space under the epidermis, wherein the plant-derived collagen comprises rh collagen.

9. A method of filling the tissue space under the epidermis, wherein the plant-derived collagen is obtained from a genetically modified plant.

10. A method of filling the tissue space under the epidermis, wherein the genetically modified plant is genetically selected from tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. Modified plant, method.

11. A method of filling the tissue space under the epidermis, wherein the genetically modified plant is a tobacco plant.

12. A method of filling the tissue space under the epidermis, wherein the genetically modified plant is of human deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-alpha, P4H-beta, and LH3. A method comprising an expressive sequence of at least one gene sequence.

13. A method of filling the tissue space under the epidermis, wherein the plant-derived human collagen is at least one modified human collagen alpha-1 as shown in SEQ ID NO: 3 and expressed in a genetically modified plant. chain; And at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO: 6 and expressed in a genetically modified plant; Wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H).

14. A method of filling the tissue space under the epidermis, the method further comprising expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C.

15. A method of filling the tissue space under the epidermis, wherein the human collagen alpha-1 chain is encoded by a sequence as set forth in SEQ ID NO: 1.

16. A method of filling the tissue space under the epidermis, wherein the human collagen alpha-2 chain is encoded by a sequence as set forth in SEQ ID NO: 2.

17. A method of filling the tissue space below the epidermis, wherein the exogenous P4H is mammalian P4H.

18. A method of filling a tissue space under the epidermis, wherein the exogenous P4H is human P4H.

19. A method of filling the tissue space under the epidermis, the method further comprising targeting human collagen alpha-1 to the vacuoles of a plant or genetically modified plant, and degrading it to ficin.

20. A method of filling the tissue space under the epidermis, the method further comprising targeting human collagen alpha-2 to the vacuoles of a plant or genetically modified plant, and degrading it to a ficin.

21. A method of filling a tissue space under the epidermis, wherein the plant-derived human collagen is atelocollagen.

22. A method of filling a tissue space under the epidermis, wherein the light source comprises a light emitting diode (LED), a laser, or a xenon lamp.

23. A method of filling a tissue space under the epidermis, wherein the photoinitiator reacts to visible light to induce polymerization of a polymerizable solution.

24. A method of filling a tissue space under the epidermis, wherein the visible light has a wavelength of 390 nm to 700 nm.

25. A method of filling the tissue space under the epidermis, wherein the photoinitiator is selected from the group consisting of eosin Y+ triethanolamine or riboflavin.

26. A method of filling the tissue space under the epidermis, wherein the photoinitiator reacts with ultraviolet (uv) to induce polymerization of a polymerizable solution.

27. A method of filling the tissue space under the epidermis, wherein the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4 2-hydroxy-1-[4-( 2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (IRGACURE® 2959).

28. A method of filling a tissue space under the epidermis, wherein the photoinitiator reacts to infrared light to induce polymerization of a polymerizable solution.

29. A method of filling the tissue space below the epidermis, wherein the polymerizable solution is introduced into the tissue space through a hollow needle or cannula ranging from 27-gauge to 33-gauge.

30. A method of filling a tissue space under the epidermis, wherein the polymerizable solution in the tissue space is shaped or shaped into the desired arrangement through manual massage.

31. A method of filling a tissue space under the epidermis, wherein the polymerizable solution in the tissue space is shaped or shaped into a desired arrangement through a shaping or shaping mechanism.

32. A method of filling a tissue space below the epidermis, wherein the polymerizable solution in the tissue space is essentially non-gelable at room temperature.

33. A method of filling a tissue space under the epidermis, wherein the polymerizable solution in the tissue space is essentially non-gelable at 37°C.

34. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. Having a reduced viscosity at room temperature.

35. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. With a reduced viscosity at 37°C.

36. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. The method being introduced into the tissue space with a reduced force at room temperature.

37. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising human or bovine collagen tissue extracted at the same concentration and formulation. And introduced into the tissue space with a reduced force at °C.

38. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. With increased tissue strengthening.

39. Use of a polymeric solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein the polymeric solution is crosslinkable, plant-derived human collagen and prior to application of visible light Or at the same time forming or shaping the polymerizable solution into a desired configuration to reduce wrinkles, folds, fine lines, wrinkles, or scars, comprising a photoinitiator to induce polymerization.

40. The use of a polymerizable solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein the crosslinkable, plant-derived human collagen is methacrylated or thiolated To become, use.

41. The use of a polymeric solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein the polymerizable solution is hyaluronic acid (HA) or a modified derivative thereof, Poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modification thereof A derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

42. The use of a polymerizable solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene Glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) The modified derivative includes a photopolymerizable modified derivative.

43. Use of a polymerizable solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene Glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) The use of modified derivatives comprising methacrylated derivatives or thiolated derivatives.

44. A method of filling the tissue space under the epidermis, the method comprising:

Introducing a polymerizable solution into the tissue space, wherein the polymerizable solution comprises crosslinkable, plant-derived human collagen.

45. A method of filling the tissue space under the epidermis, the method comprising:

(a) shaping or shaping the polymerizable solution into a desired arrangement within the tissue space.

46. A method of filling a tissue space under the epidermis, wherein the shaping or shaping step reduces wrinkles, folds, fine lines, wrinkles or scars.

47. As a method of filling the tissue space under the epidermis, the polymerizable solution is hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modification thereof. Derivatives, oxidized cellulose (OC) or modified derivatives thereof, polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) Or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

48. A method of filling a tissue space under the epidermis, wherein the plant-derived collagen comprises rh collagen.

49. A method of filling a tissue space under the epidermis, wherein the plant-derived collagen is obtained from a genetically modified plant.

50. A method of filling the tissue space under the epidermis, wherein the genetically modified plant is genetically selected from tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. Modified plant, method.

51. A method of filling the tissue space under the epidermis, wherein the genetically modified plant is a tobacco plant.

52. A method of filling the tissue space under the epidermis, wherein the genetically modified plant is of human deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-alpha, P4H-beta, and LH3. A method comprising an expressive sequence of at least one gene sequence.

53. A method of filling the tissue space under the epidermis, wherein the plant-derived human collagen is at least one modified human collagen alpha-1 as shown in SEQ ID NO: 3 and expressed in a genetically modified plant. chain; And at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO: 6 and expressed in a genetically modified plant; Wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H).

54. A method of filling the tissue space under the epidermis, further comprising expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C.

55. A method of filling the tissue space under the epidermis, wherein the human collagen alpha-1 chain is encoded by a sequence as set forth in SEQ ID NO: 1.

56. A method of filling the tissue space under the epidermis, wherein the human collagen alpha-2 chain is encoded by a sequence as set forth in SEQ ID NO: 2.

57. A method of filling the tissue space below the epidermis, wherein the exogenous P4H is mammalian P4H.

58. A method of filling the tissue space below the epidermis, wherein the exogenous P4H is human P4H.

59. A method of filling the tissue space under the epidermis, further comprising targeting human collagen alpha-1 to the vacuoles of a plant or genetically modified plant, and degrading it to ficin.

60. A method of filling the tissue space under the epidermis, the method further comprising targeting human collagen alpha-2 to the vacuoles of a plant or genetically modified plant, and degrading it to ficin.

61. A method of filling a tissue space under the epidermis, wherein the plant-derived human collagen is atelocollagen.

62. A method of filling a tissue space below the epidermis, wherein the polymerizable solution is introduced into the tissue space through a hollow needle or cannula ranging from 27-gauge to 33-gauge.

63. A method of filling a tissue space under the epidermis, wherein the polymerizable solution in the tissue space is shaped or shaped into the desired arrangement through manual massage.

64. A method of filling a tissue space under the epidermis, wherein the polymerizable solution in the tissue space is shaped or shaped into a desired arrangement through a shaping or shaping mechanism.

65. A method of filling a tissue space below the epidermis, wherein the polymerizable solution in the tissue space is essentially non-gelable at room temperature.

66. A method of filling a tissue space below the epidermis, wherein the polymerizable solution in the tissue space is essentially non-gelable at 37°C.

67. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. Having a reduced viscosity at room temperature.

68. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. With a reduced viscosity at 37°C.

69. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. The method being introduced into the tissue space with a reduced force at room temperature.

70. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising human or bovine collagen tissue extracted at the same concentration and formulation. And introduced into the tissue space with a reduced force at °C.

71. A method of filling the tissue space under the epidermis, wherein a polymerizable solution comprising plant-derived human collagen is compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. With increased tissue strengthening.

72. Use of a polymeric solution injected into the tissue space under the epidermis to reduce wrinkles, folds, fine lines, wrinkles, or scars, wherein the polymerizable solution comprises crosslinkable, plant-derived human collagen, and Use of shaping or shaping the polymerizable solution into a desired configuration to reduce folds, fine lines, wrinkles, or scars.

73. A method of inducing a cellular growth scaffold in a tissue space under the epidermis, the method comprising introducing a solution into the tissue space, the solution comprising:

(a) plant-derived human collagen; And

(b) at least one growth factor or source thereof.

74. A method of inducing a cellular growth scaffold in a tissue space below the epidermis, wherein the source of the at least one growth factor comprises plasma or platelet-rich plasma.

75. A method of inducing a cellular growth scaffold in a tissue space below the epidermis, wherein the cellular growth scaffold promotes healing or replacement due to degradation or injury of the collagen-comprising tissue.

76. A method of inducing a cellular growth scaffold in the tissue space under the epidermis, wherein the collagen-containing tissue is a group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones or teeth. Selected from, method.

77. A method of inducing a cellular growth scaffold in a tissue space below the epidermis, wherein the cellular growth scaffold promotes healing of tendinitis.

78. A method of inducing a cellular growth scaffold in a tissue space below the epidermis, wherein the plant-derived collagen comprises rh collagen.

79. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the plant-derived collagen is obtained from a genetically modified plant.

80. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the genetically modified plant is tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and A genetically modified plant selected from cotton.

81. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the genetically modified plant is a tobacco plant.

82. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the genetically modified plant is a human deoxy selected from the group consisting of COL1, COL2, P4H-alpha, P4H-beta, and LH3. A method comprising an expressive sequence of at least one gene sequence of ribonucleic acid (DNA).

83. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the plant-derived human collagen is as set forth in SEQ ID NO: 3 and at least a modified one as expressed in a genetically modified plant Human collagen alpha-1 chain of; And at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO: 6 and expressed in a genetically modified plant; Wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H).

84. A method of inducing a cellular growth scaffold in the tissue space under the epidermis, further comprising the step of expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. Included as, method.

85. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the human collagen alpha-1 chain is encoded by a sequence as set forth in SEQ ID NO: 1.

86. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the human collagen alpha-2 chain is encoded by a sequence as set forth in SEQ ID NO: 2.

87. A method of inducing a cellular growth scaffold in a tissue space below the epidermis, wherein the exogenous P4H is mammalian P4H.

88. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein the exogenous P4H is human P4H.

89. A method of inducing a cellular growth scaffold in the tissue space under the epidermis, further comprising targeting human collagen alpha-1 to the vacuoles of a plant or genetically modified plant, and decomposing it to ficin, Way.

90. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, further comprising targeting human collagen alpha-2 to the vacuoles of a plant or genetically modified plant, and decomposing it into ficin, Way.

91. A method of inducing a cellular growth scaffold in a tissue space below the epidermis, wherein the plant-derived human collagen is atelocollagen.

92. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein a solution comprising plant-derived human collagen is a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. Compared with a reduced viscosity at room temperature.

93. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein a solution comprising plant-derived human collagen is a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. With a reduced viscosity at 37° C. in comparison.

94. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein a solution comprising plant-derived human collagen is a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. Compared with a reduced force at room temperature, introduced into the tissue space.

95. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein a solution comprising plant-derived human collagen is a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. Compared to the tissue space at 37° C. with reduced force.

96. A method of inducing a cellular growth scaffold in the tissue space below the epidermis, wherein a solution comprising plant-derived human collagen is a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. A method having an increased scaffolding formation in comparison or promoting an increase in cell growth.

97. Use of a solution injected into the tissue space below the epidermis to induce a cellular growth scaffold, the solution being plant-derived human collagen to promote healing or replacement due to degradation or injury of collagen-containing tissue. And at least one growth factor or source thereof.

98. Use of a solution injected into the tissue space below the epidermis to induce a cellular growth scaffold, wherein the source of at least one growth factor comprises plasma or platelet-rich plasma.

Justice

As used herein, the singular forms (“a”, “an” and “the”) include plural references unless the context clearly indicates otherwise. For example, the term “molecule” also includes a plurality of molecules.

As used herein, the term “about” refers to ± 10% or ± 5%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugations include “including, but not limited to”. it means.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure is an additional component, only if the additional component, step and/or portion does not substantially alter the basic and novel characteristics of the claimed composition, method or structure, It means that it can contain steps and/or parts.

As used herein, the term “method” refers to a given task or manner known by the practitioner, including, but not limited to, methods, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine Refers to the methods, means, techniques and procedures for achieving a given task that is easily developed from means, techniques and procedures.

As used herein, the phrase “genetically modified plant” refers to any lower (eg, moss) or higher (related) plant or their (eg, moss) that is stably or transiently transformed with an exogenous polynucleotide sequence. For example, a cell suspension) of tissue or isolated cells. Examples of plants include, but are not limited to, tobacco, corn, alfalfa, rice, potatoes, soybeans, tomatoes, wheat, barley, canola, cotton, carrots, as well as lower plants such as moss.

As used herein, the phrase “collagen chain” encompasses collagen subunits, such as the alpha 1 or alpha 2 chain of collagen fibers, preferably type I fibers. As used herein, the phrase “collagen” refers to an assembled collagen trimer comprising two alpha 1 chains and one alpha 2 chain for type I collagen. Collagen fibers are collagen without terminal propeptides C and N.

As used herein, the phrase “telopeptide-comprising collagen” encompasses soluble collagen molecules comprising telopeptides that are longer than the telopeptide residues contained in atelocollagen. Therefore, the telopeptides-comprising collagen may be a procollagen comprising a full-length propeptide. Alternatively, the telopeptide-comprising collagen may be a procollagen molecule comprising a partially degraded propeptide. More alternatively, the telopeptide-comprising collagen may be telocollagen.

As used herein, the term “procollagen” encompasses a collagen molecule (eg, human) comprising an N-terminal propeptide, a C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are set forth in SEQ ID NOs: 1, 2, 7 and 8 .

As used herein, the term “telocollagen” encompasses collagen molecules that are typically included in procollagen, but lack both the N-terminal and C-terminal propeptides which still contain telopeptides. The telopeptides of fibrotic collagen are the residues of the N-terminal propeptide and C-terminal propeptide after degradation by native N/C proteinase. Recombinant human telocollagen can be produced in cells that have been transformed to express both the exogenous human procollagen and the respective protease (ie, C or N or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NO: 10 (protease C) and SEQ ID NO: 11 (protease N). These proteases can be expressed such that these proteases accumulate in the same subcellular compartment as the collagen chain, as further described herein below.

As used herein, the term "atelocollagen" is devoid of both the N-terminal and C-terminal propeptides and at least a portion of their telopeptides, typically included in procollagens, but under suitable conditions it is It encompasses a collagen molecule that contains a sufficient portion of its telopeptides to form fibrils. Any type of atelocollagen can be produced according to the method of the present invention. Examples include fibril-forming collagen (type I, II, III, V and XI), network-forming collagen (IV, VIII and X), and collagen associated with the fibrillar surface (type IX, XII). Type and type XIV), collagen (type XIII and type XVII) generated as a transmembrane protein, or form 11-nm periodic bead-type filaments (type VI). According to one embodiment, the atelocollagen comprises the alpha 1 and/or alpha 2 chain of type I collagen.

It will be appreciated that the dermal fillers disclosed herein may, in some embodiments, comprise a genetically modified form of collagen/atelocollagen—eg, collagenase-resistant collagen, and the like.

As used herein, the phrase “plant promoter” or “promoter” includes a promoter capable of directing gene expression in cells (including DNA-containing organelles) of plants, fungi and yeast. Such promoters can be derived from plant, bacterial, viral, fungal or animal origin. Such promoters can be constitutive, i.e., can direct high levels of gene expression in a plurality of tissues, or such promoters can be tissue specific, i.e., can direct gene expression in specific tissues or tissues, or Such promoters can be inducible, i.e., can direct gene expression under stimulation, or such promoters can be chimeric, i.e., can be formed as part of at least two different promoters.

As used herein, the phrase “subcellular compartment without endogenous P4H activity” refers to any compartmentalized region of a cell that does not contain plant P4H or an enzyme with plant-like P4H activity. Examples of such subcellular compartments include vacuoles, apoplasts and cytoplasm, as well as organelles such as chloroplasts, mitochondria, and the like.

Throughout this application, the phrase “building material” encompasses the phrases “uncured building material” or “uncured building material formulation” and collectively refers to the material used to sequentially form the layers as described herein. Write. These phrases include the uncured material that forms the final object, i.e., one or more uncured modeling material formulation(s), and optionally also the uncured material, i.e., uncured, used to form the support. Includes support material formulations. The uncured building material may comprise one or more modeling agents, and may be dispensed such that different parts of the object are prepared upon curing of different modeling agents, thus with different cured modeling materials or different mixtures of cured modeling materials. Is manufactured.

As used herein, “bioprinting” refers to one or more bio-inks comprising biological components via a method compatible with an automated or semi-automated, computer-aided, additive manufacturing system as described herein. Means performing an additive manufacturing process while using the formulation(s).

Throughout this application, in the context of bioprinting, the term “object” describes the final product of additive manufacturing comprising at least a portion of a biological component. This term refers to the product obtained by the bioprinting method as described herein after removal of the support material, if it is used as part of the uncured building material. In some embodiments, the biological component comprises recombinant human collagen as described in, for example, WO 2006/035442, WO 2009/053985, and patents and patent applications derived therefrom, all of which are presented herein in whole It is included by reference as if it were.

As used throughout this application, the term “object” refers to an entire object or part thereof.

Throughout this application, a “curable material” is a compound (monomeric or oligomeric or polymeric compound) that, upon exposure to curing conditions as described herein, solidifies or hardens to form a cured molding material as described herein. Curable materials are typically polymeric materials that undergo polymerization and/or crosslinking upon exposure to a combined energy source. Alternatively, the curable material is a heat-reactive material that solidifies or hardens upon exposure to temperature changes (eg, heating or cooling). Optionally, the curable material is a biological material that undergoes a reaction to form a hardened or solid material upon a biological reaction (eg, an enzymatic-catalyzed reaction).

“Cure conditions” encompasses curing energy (eg, temperature, radiation) and/or substances or reagents that promote curing.

In some of any of the embodiments described herein, the curable material is a photopolymerizable material that polymerizes or undergoes crosslinking upon exposure to radiation as described herein, and in some embodiments, the curable material is subjected to UV-vis radiation as described herein. It is a UV-curable or visible light-curable material that polymerizes or undergoes crosslinking upon exposure.

In some of any of the embodiments described herein, the curable material may be a monomer, oligomer, or short chain polymer, each polymerizable as described herein.

As used herein, the term “curable” encompasses the terms “polymerizable” and “crosslinkable”.

As used herein, "aeroponics" is the process of growing plants in an air or humid environment without the use of soil or aggregate media (known as "geoponics").

As used herein, “hydroponics” is the process of growing plants without soil (“agroculture”) using a solution of mineral nutrients in a water solvent.

As used herein, "endosphere" includes all endophytes of plants.

As used herein, “exudate” is a fluid that is excreted by an organism through a pore or wound. "Exudate" is the process of releasing "exudate".

As used herein, "hydroponic cultivation" is the process of growing plants without soil using a solution of mineral nutrients in a water solvent ("agroculture").

As used herein, “integression” or “introgression hybridization” refers to the gene of one species through repeated backcrossing of an interspecific hybrid with one of the parent species, as opposed to simple hybridization. It is the movement of genes from the pool into another species' gene pool, resulting in a complex mix of parental genes (ie, "gene flow").

As used herein, a “metabolome” refers to a complete small molecule chemical substance found within a “biological sample” (but not limited to a cell, organelle, organ, tissue, tissue extract, biofluid, or organism). It is a set. The small molecule chemicals of the metabolite may be “endogenous metabolites” or “exogenous metabolites”. “Endogenous metabolites” are naturally produced by the organism and include, but are not limited to, amino acids, organic acids, nucleic acids, fatty acids, amines, sugars, vitamins, cofactors, pigments and antibiotics. "Exogenous chemicals" are not naturally produced by the organism and include, but are not limited to, drugs, environmental pollutants, food additives, toxins and other xenobiotics. "Endogenous metabolites" consist of endogenous metabolites, while "exogenous metabolites" consist of "exogenous chemicals". "Endogenous metabolites" consist of "primary metabolites" and "secondary metabolites", particularly with respect to plants, fungi and prokaryotes. “Primary metabolites” consist of “primary metabolites” (ie, metabolites directly involved in the normal growth, development and reproduction of an organism), while “second metabolites” refer to “second metabolites” ( That is, metabolites that are not directly involved in the normal growth, development or reproduction of an organism). Secondary metabolites often have significant ecological functions.

As used herein, a “metabolite” is a low molecule, usually having a molecular weight of less than 1,500 Da. "Metabolites" may include, but are not limited to, glycolipids, polysaccharides, short peptides, small oligonucleotides, organic acids, taxanes, alkaloids and strigolactones, while very large macromolecules (eg, Proteins, mRNA, rRNA and DNA) are generally not metabolites and are not part of a metabolite.

As used herein, “SILVA database” is the SILVA ribosomal RNA database.

All samples obtained from an organism, including those subject to further processing of any kind, are considered to be obtained from that organism.

Methods of DNA isolation, sequencing, amplification, and/or cloning are known to those of skill in the art. The most commonly used method for DNA amplification is PCR (polymerase chain reaction; for example, PCR Basics: from background to Bench, Springer Verlag, 2000); Eckert et al., 1991. PCR Methods and Applications 1: 17]). Additional suitable amplification methods include ligase chain reaction (LCR), transcriptional amplification and self-sustaining sequence replication, and nucleic acid-based sequence amplification (NASBA). Likewise, methods for RNA and protein isolation, characterization, and the like and methods of protein expression are known to those skilled in the art.

The following examples are presented to more fully illustrate some embodiments of the dermal fillers and uses thereof disclosed herein. However, this should in no way be construed as limiting the broad scope of the dermal fillers disclosed herein or their uses. Those skilled in the art can easily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

Example

Example 1. Construct and transformation scheme.

The construction of expression cassettes and vectors used in this study is illustrated in Figures 1A-1D (see US Pat. No. 8,455,717). In this study, all coding sequences were optimized for expression in tobacco and chemically synthesized to have the desired flanking regions ( SEQ ID NO: 1, 4, 7, 12, 14, 16, 18, 20, 22 ). 1A: Synthetic genes encoding Col1 and Col2 ( SEQ ID NO: 1, 4 ) fused to or without a signal fused to a vacuole signal or an apoplast signal (encoded by SEQ ID NO: 7 ) and 5 'UTR ( SEQ ID NO: 10 ) and chrysanthemum rbcS1 3'UTR and terminator ( SEQ ID NO: 11 ) were cloned in an expression cassette. The complete expression cassette was cloned into multiple cloning sites of the pBINPLUS plant transformation vector (van Engelen et al., 1995, Transgenic Res 4: 288-290). 1B: Synthetic gene encoding P4H beta-human, P4H alpha-human and P4H-plant fused to or without signal to vacuole signal or to apoplast signal (encoded by SEQ ID NO: 7 ) ( SEQ ID NO. : 12, 14 and 16 ) were cloned in an expression cassette consisting of the CaMV 35S promoter and TMV omega sequence and Agrobacterium nopaline synthetase (NOS) terminator carried by the vector pJD330 (Galili et al., 1987 , Nucleic Acids Res 15:3257-3273]). The complete expression cassette was cloned into multiple cloning sites of the pBINPLUS vector carrying the expression cassette of Col1 or Col2. Figure 1C: Synthetic gene encoding proteinase C and proteinase N fused to vacuole signal or to apoplast signal (encoded by SEQ ID NO: 7 ) ( SEQ ID NO: 18, 20 ) Was cloned in an expression cassette consisting of a chrysanthemum rbcS1 promoter and 5'UTR ( SEQ ID NO: 10 ) and a chrysanthemum rbcS1 3'UTR and terminator ( SEQ ID NO: 11 ). The complete expression cassette was cloned into the multiple cloning site of the pBINPLUS plant transformation vector. Figure 1D: Strawberry vein banding virus (SVBV) promoter (NCBI deposit AF331666 REGION: 623.950) fused to the vacuole signal or to the apoplast signal (encoded by SEQ ID NO: 7 ) or without a signal. version AF331666.1 GI: 13345788), and the Agrobacterium tumefaciens octopine synthase (OCS) terminator (NCBI Accession Z37515 REGION: 1344.1538 version Z37515.1 GI: synthetic gene encoding LH3 is terminated by 886843) (SEQ ID NO: 22 ) was cloned into the multiple cloning site of the pBINPLUS vector carrying the expression cassettes of Col1 and P4H beta.

A co-transformation scheme using the expression cassettes described in FIGS. 1A-1D into host plants is illustrated in FIG. 2 . Each expression cassette insert is indicated by the short name of the coding sequence. The coding sequence and related SEQ ID NO . are set forth in Table 1. Each co-transformation is performed by two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector carrying 1, 2 or 3 expression cassettes. Promoters and terminators are specified in Figures 1A-1D .

Example 2. Plant collagen expression.

Synthetic polynucleotide sequences encoding the proteins listed in Table 1 below were designed and optimized for expression in tobacco plants.

Signal peptide

1. The vacuole signal sequence of the barley gene for thiol protease aleurain precursor (NCBI deposit P05167 GI:113603) MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA ( SEQ ID NO: 24 ).

2. Apoplast signaling of Arabidopsis thaliana endo-1,4-beta-glucanase (Cell, NCBI deposit CAA67156.1 GI:2440033); SEQ ID NO. SEQ ID NO, encoded by 7 . 9.

Construction of the plasmid

Plant expression vectors were constructed as taught in Example 1, and the composition of each constructed expression vector was confirmed through restriction analysis and sequencing.

An expression vector comprising the following expression cassette was constructed:

1. Collagen Alpha 1

2. Collagen alpha 1 + human P4H beta subunit

3. Collagen alpha 1 + human P4H beta subunit + human LH3

4. Collagen Alpha 2

5. Collagen alpha 2+ human P4H alpha subunit

6. Collagen Alpha 2+ Arabidopsis P4H

7. Human P4H beta subunit + human LH3

8. Human P4H alpha subunit

Translationally fusing each of the above-described coding sequences to a vacuole transport peptide or to an apoptoplasmic transport peptide, or without any transport peptide sequence, in which case cytoplasmic accumulation is expected.

Plant transformation and PCR screening

Tobacco plants (Nicotiana tabacum, Samsun NN) were transformed with the expression vector described above according to the transformation scheme taught in FIG. 2 .

The resulting transgenic plants were screened through multiplex PCR using four primers designed to amplify a 324 bp fragment of collagen alpha 1 and a 537 bp fragment of collagen alpha 2 (Table 2). 3 illustrates the results of one multiplex PCR screen.

Example 3. Detection of human collagen in transgenic tobacco plants.

Tobacco Transformant 2, by grinding 500 mg of leaves with a "complete" protease inhibitor cocktail (product #1836145 from Roche Diagnostics GmbH, 1 tablet in 50 ml buffer) at 0.5 ml 50 mM Tris-HCl pH=7.5 Total soluble protein was extracted from 3 and 4. The crude extract was mixed with 250 μl 4X. In sample application buffer containing 10% beta-mercapto-ethanol and 8% SDS, the samples were heated for 7 minutes and centrifuged at 13,000 rpm for 8 minutes. 20 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested with an anti-collagen I (denatured) antibody (#AB745 from Chemicon Inc.) in a standard Western blot procedure ( FIG. 4 ). WT is a wild type tobacco. Positive collagen bands are visible in plants that are PCR positive for collagen type I alpha 1 or alpha 2 or both. A positive control band of 500 ng collagen type I from human placenta (#CC050 from Chemicon Inc.) represents about 0.3% of the total soluble protein (about 150 μg) in samples from transgenic plants.

When collagen is targeted to the vacuoles, plants expressing collagen at an expected molecular weight of about 1% or less of total soluble protein were detected ( FIG. 4 ). Subcellular targeting of full-length collagen to Apoplast was successfully achieved ( FIG. 5 ). Plants expressing collagen in the cytoplasm (ie, no targeting peptide) did not accumulate collagen to detectable levels, indicating that subcellular targeting of collagen in plants is critical to success.

In addition, in contrast to the studies of Ruggiero et al. 2000 and Merle et al. 2002, which showed that collagen lacking N-propeptide received significant proteolysis, using this approach, with C-propeptide and N-propeptide Full-length collagen protein accumulated at high levels in the subcellular compartment.

The present data also shows that the crossing of two plants, each expressing a different collagen chain type, allows the selection of plants in which they express the optimal level of each chain type and subsequently plants to achieve the desired collagen producing plants. It clearly shows the advantage in that it enables mating.

The collagen produced by the plants of the present invention contains a native propeptide and is, therefore, expected to form a larger protein than a human control purified by proteolysis. The calculated molecular weights of collagen alpha 1 and alpha 2 chains without hydroxylation or glycosylation are as follows: Col1--136 kDa without propeptide, Col1--95 kDa without propeptide, Col2--127 with propeptide kDa, Col2--92 kDa without propeptide.

As can be seen in Figure 4 , the Col1 band in transformants 3-5 and 3-49 appears to be larger than the Col1 band in other plants. It is co-expressed in these plants and is collagen by human proline-4-hydroxylase complete enzyme consisting of alpha subunits and beta subunits targeted to the same subcellular compartments (e.g. vacuoles) as human collagen chains. Represents proline hydroxylation in the chain.

Example 4. Collagen triple helix assembly and thermal stability in transgenic plants.

Assembly and helix thermal stability of the collagen triple helix in transgenic plants were tested by thermal denaturation followed by trypsin or pepsin digestion of the total crude protein extract of the transgenic plants ( FIGS. 6A to 6B ).

In the first experiment, 500 mg of leaves were crushed in 0.5 ml 50 mM Tris-HCl pH=7.5, centrifuged at 13,000 rpm for 10 minutes, and the supernatant was collected, thereby expressing tobacco 2-9 (col alpha 1 only and P4H. Not expressed) and tobacco 3-5 (expressing both col alpha1+2 and P4H) total soluble protein was extracted. 0 μl of the supernatant was heat treated (15 min at 33°C or 43°C), after which it was immediately placed on ice. Trypsin digestion was initiated by adding 1 mg/ml trypsin of 6.mu.l of each sample to 50 mM Tris-HCl pH=7.5. Samples were incubated at room temperature (about 22° C.) for 20 minutes. Digestion was terminated by the addition of 20 μl 4X sample application buffer containing 10% betamercaptoethanol and 8% SDS, and the sample was heated for 7 minutes and centrifuged at 13,000 rpm for 7 minutes. 50 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested with anti-collagen I antibody (#AB745 from Chemicon Inc.) using standard Western blot procedures. A positive control was a sample of 500 ng human collagen I (#CC050 from Chemicon Inc., extracted from human placenta by pepsin digestion), which was w.t. 50 μl of total soluble protein extracted from tobacco was added.

As shown in Figure 6a , the collagen triple helix formed in plant #3-5 as well as the control human collagen were resistant to denaturation at 33°C. In contrast, collagen formed by plant #2-9 was denatured at 33°C. This difference in thermal stability indicates successful triple helix assembly and post-translational proline hydroxylation in transformants #3-5 expressing both collagen alpha 1 and collagen alpha 2, as well as P4H beta and alpha subunits. .

The two bands in transformant #2-9 can represent a stable dimer or trimer after 7 minutes of heating with SDS and mercaptoethanol. Similar bands are visible in human collagen (top panel) and transformants #3-5. A possible explanation is the covalent bond (crosslinking) between two peptides in different triple helices formed after oxidative deamination of two lysines by lysine oxidase.

In a second experiment, 500 mg of leaves were crushed in 0.5 ml of 100 mM Tris-HCl pH=7.5 and 300 mM NaCl, centrifuged at 10,000 rpm for 7 minutes, and the supernatant was collected to obtain tobacco 13-6 (collagen I alpha And total soluble protein from expressing the alpha 2 chain—indicated by arrows, expressing human P4H alpha and beta subunits and human LH3). 50 μl of the supernatant was heat treated (20 min at 33° C., 38° C. or 42° C.) and then immediately placed on ice. Pepsin digestion was initiated by adding 4.5 μl of 0.1M HCl and 4 μl of 2.5 mg/ml pepsin in 10 mM acetic acid to each sample. Samples were incubated for 30 minutes at room temperature (about 22° C.). The digestion was terminated by adding 5 μl of unbuffered 1 M Tris. Each sample was mixed with 22 μl 4X sample application buffer containing 10% beta-mercapto-ethanol and 8% SDS, and the samples were heated for 7 minutes and centrifuged at 13,000 rpm for 7 minutes. 40 μl of the supernatant was loaded on a 10% polyacrylamide gel and tested with anti-collagen I antibody (#AB745 from Chemicon Inc.) in a standard Western blot procedure. The positive control was w.t. It was a sample of about 50 ng human collagen I (#CC050 from Chemicon Inc., extracted from human placenta by pepsin digestion) added to the total soluble protein from tobacco.

As illustrated in FIG . 6B , the collagen triple helix formed in plant #13-6 was resistant to denaturation at 42°C. The cleavage of the propeptide is first visible at 33° C., and when the temperature rises to 38° C. and again up to 42° C., the efficiency gradually increases. The cleaved collagen triple helix domain shows a migration on a gel similar to that of pepsin treated human collagen. The human collagen used in this experiment was extracted from the human placenta by pepsin proteolysis, and thus lacks propeptide and some telopeptides.

Example 5. Plant P4H expression.

Induction of P4H in native plants

Tobacco P4H cDNA was cloned and used as a probe to determine conditions and treatments that would induce endogenous P4H expression. Northern blot analysis ( FIG. 7 ) clearly shows that P4H is expressed at a relatively high level at the growth point and at a low level at the leaf. P4H levels were significantly induced in the leaves 4 hours after abrasion treatment ("wound" in the bottom panel). Similar results were achieved using other stress conditions (not shown).

Detection of human P4H alpha and beta subunits and collagen alpha 1 and alpha 2 chains in transgenic tobacco plants

Anti-human P4H alpha subunit antibody (#63-163 from ICN Biomedicals Inc.), anti-human P4H beta subunit antibody (#NMAB2701 from Chemicon Inc.) and anti-collagen I antibody (from Chemicon Inc.) #AB745) was used to detect human P4H alpha and beta subunits and collagen type I alpha 1 and alpha 2 chains in transgenic tobacco plants. The results of Western blot probed with these antibodies are presented in Figure 8 .

Expression of the P4H alpha, P4H beta and collagen 1 alpha 1 and alpha 2 bands was confirmed in plants 13-6 (also transformed with human LH3). The calculated molecular weights of P4H alpha and beta including the vacuole signal peptide are 65.5 kDa and 53.4 kDa, respectively. The calculated molecular weights of the collagen alpha 1 and alpha 2 chains without hydroxylation or glycosylation and with propeptide are 136 kDa and 127 kDa, respectively.

Example 6. Vacuole-targeted collagen is stably expressed in dark-growing plants.

Plants expressing collagen:

A 20-279 parental tobacco plant line was generated by co-transformation with an expression vector expressing P4Hbeta+LH3 and another expression vector expressing P4Halpha. Each gene is present following the vacuole targeting determinant of allurein, plant vacuole thiol protease.

2-300 parental tobacco plant lines were generated by co-transformation with an expression vector expressing col1 and another expression vector expressing col2. Each gene is present following the vacuole targeting determinant of allurein, plant vacuole thiol protease.

13-652 plants were generated by co-transformation of tobacco plants with an expression vector encoding Col1, P4Hbeta and LH3 and a second expression vector encoding Col2 and P4H alpha. Each gene is present following the vacuole targeting determinant of allurein and plant vacuole thiol protease, and the cassette sequence included in the vector is described in Example 1 above.

Contrast test

Analysis of six 13-6/52 homozygous plants. Samples from leaf #4+5/6 were taken daily for 8 days at the same time (12:30), from 3 plants grown in regular conditions (16 hours under light conditions and 8 hours under dark conditions) and only in the dark. It was harvested from three grown plants.

Total protein extraction and western blot analysis

90 mg of tobacco leaves were homogenized with mixer mill type MM301 (Retsch) in extraction buffer (100 mM Tris HCl pH=7.5 available from Roche catalog number, 04-693-116-001, protease inhibitor cocktail) at 4°C. . After 30 minutes of centrifugation (20,000Xg at 4°C), the supernatant was collected. Protein samples were fractionated on 8% SDS-PAGE (Laemmli 1970) and transferred to a nitrocellulose membrane using a BIO-RAD ^™ protein TRANS-BLOT ^™ instrument. The membrane was blocked in 3% (g/v) skim milk (Difco) at room temperature for 30 minutes, and then reacted with a commercial rabbit anti-human collagen type I polyclonal antibody (Chemicon) overnight (on) at room temperature. The membrane was rinsed 3 to 5 times with water and then washed in TBS for 30 minutes. After incubation with a secondary antibody (goat anti-rabbit-IgG antibody (Chemicon) conjugated to alkaline phosphatase (AP)) at room temperature for 2 hours, the membrane was rinsed 3 to 5 times with water and washed in TBS for 30 minutes . Immunodetection was performed with nitrotetrazolium blue chloride (NBT, Sigma) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP, Sigma) at room temperature for 2 hours to on.

result

As shown in Figure 9 , tobacco plants that are transgenic for vacuole-targeted collagen express pro-alpha-1 and pro-alpha-2 (lane 1). Collagen from dark grown vacuole targeted plants showed similar stability (lane 2), demonstrating the unusual stability of collagen produced according to the teachings of the present invention.

Examples 7 to 13.

Common materials and methods

Collagen Extraction and Enzymatic Reaction: In a blender, 300 g of tobacco leaves, 5 g PVPP and 2 g of activated carbon supplemented cold extraction buffer (360 mg potassium-meta-bisulfite, 530 mg L-cysteine and 1 g EDTA). Containing, 600 ml of 100 mM Tris-HCl pH 7.5) (see also US Pat. No. 8,759,487). Blending was performed for 5 minutes at 1-minute intervals to keep the temperature below 15°C. The crude extract was filtered through a gauze pad and centrifuged at 25,000 g, 5° C. for 30 minutes. Collecting the supernatant; CaCl ₂ was added to a final concentration of 10 mM. The supernatant was partitioned into 10 ml samples. The desired enzyme was added to each 10 ml sample according to the conditions set forth in Table 3 below.

Enzyme Description: Ficin from Peak Tree Latex (Sigma, Cat. #F4125), Subtilisin from Bacillus licheniformis (Sigma, Cat. #P5459-5gr), Bromelain from Pineapple Stem (Sigma, Catalog #B4882-10gr), papain from Carica papaya (Fluka, catalog #76220-25gr), sabinase 6.0 t type W (Novozymes, catalogue from alkaline bacterium Bacillus lentus) #PX92500501), Nutrase 1.5 MG from Bacterium amyloliquefaciens (Novozymes, catalog #PW201041), Protamex, commercial Bacillus proteinase complex (Novozymes, catalog #PW2A1021), Alcalase 3.0 T, Bacillus subtilis alkaline proteinase (Novozymes, catalog #PJ90000901), Esperase 6.0 T, alkaline bacterium Bacillus lentus (Novozymes, catalog # PE90110401), Alcalase 2.4 L FG, Bacillus subtilis Alkaline proteinase (Novozymes, catalog #PLN05330), Esperase 8.0 L, and alkaline bacterium Bacillus lentus (Novozymes, catalog # PE00077) were all donated by Novozymes. Trypsin, pancreatic trypsin 6.0 S type free, derived from animal pancreas (Novozymes, catalog #P245-D20). TRYPZEAN ^™ , a recombinant trypsin expressed in maize was purchased from Sigma Chemical Co. (catalog #: T3449).

Determination of Atelocollagen Concentration: The concentration of atelocollagen produced according to Examples 9 to 10 was assayed by the following two methods:

SIRCOL ^™ Assay: SIRCOL ^™ Collagen Assay Kit was purchased from Biocolor Ltd. (Cat. No. 85000). This assay is based on the interaction of Sirius Red dye with collagen triple helix. The analysis was performed according to the supplier's instruction manual, 4th edition, 2002. Using bovine collagen standards, a calibration curve (0-50 μg collagen) was prepared. Three samples of a collagen solution of 10 μl to 50 μl in 10 mM HCl were placed into a 1.5 ml Eppendorf tube and the volume was made to 100 μl with 0.5 M acetic acid. 1 ml of SIRCOL ^™ dye reagent was added to each tube, and the tube was shaken at room temperature for 30 minutes. The tube was centrifuged at 12,000 rpm for 10 minutes at room temperature, the supernatant was aspirated, and the tube was inverted onto the absorbent paper to remove the residual supernatant. Using a cotton swab, any excess droplets were removed from the wall of the tube. 1 ml of alkaline reagent was added to each tube, mixed well, and incubated for 10 minutes at room temperature. The absorbance at 540 nm was measured using a spectrophotometer, and the concentration of collagen was calculated by comparing with the calibration curve using 10 mM HCl as a blank sample.

SDS-PAGE Instant Blue Assay: SDS PAGE, before loading onto 8% acrylamide, samples were heated in SAB buffer (reduction conditions) for 5 minutes and centrifuged at 12,000 rpm for 5 minutes. The gel was run in a Mini Protean 3 unit (BioRad #165-3301, 165-3302). Instant Blue reagent (Novexin #ISB01L) was applied to the gel until the protein was visualized as a blue band on the gel. The gel was rinsed with water and dried. The concentration of collagen bands was calculated by densitometry compared to a human standard loaded on the same gel.

Coomassie Analysis: A sample of collagen (in 10 mM HCl) was titrated to pH 7.5 with 1 M Tris. Sample application buffer containing 10% beta-mercaptoethanol and 8% SDS was added by diluting 4 times in 30 μl of the pH titrated sample. The sample was heated for 7 minutes. 30 μl of the supernatant was loaded on a 10% polyacrylamide gel, and separated at 100 volts for 2 hours. The gel was transferred to Cooma's clock solution with shaking for 1 hour. Coomassie dye was removed using a standard bleaching solution.

SDS-PAGE and Western blot analysis of alpha-1 and alpha-2 collagen chains: Samples were heated in reducing sample application buffer (containing 2.5% beta-mercaptoethanol and 2% SDS) for 7 minutes, and at 13,000 rpm. Centrifuged for 15 minutes. 30 μl of the supernatant was separated on a 10% polyacrylamide gel. After separation, samples were blotted onto a nitrocellulose membrane using standard Western blot protocols. After transfer, the membrane was incubated with an anti-collagen I antibody (Chemicon Inc. catalog #AB745) for immunodetection of alpha-1 and alpha-2 collagen chains. Molecular weight markers were purchased from Fermentas Inc. (catalog #SM0671).

Control: A positive control of human skin collagen type I purchased from Calbiochem (#234138) was used as a marker for Western blot analysis. The grinding control sample reflects the pellets derived from tobacco leaves just prior to resuspension in extraction buffer. The “D” control sample reflects the same pellet after resuspension in extraction buffer. The “K” control sample contained ficin-digested procollagen in 10 mM HCl. Background To monitor picin-independent protease activity, picin-free cleavage samples were always prepared in parallel with all picin degradation tests.

Purification of collagen from transgenic plants: collagen- by addition of 30 mg/L trypsin or 5 mg/L (50 μl/L) subtilisin (Sigma #P5459) or 5 mg/L ficin (Sigma #F4125) Degradation of propeptide in the containing extract was initiated. Proteolysis was carried out at 15° C. for 4 hours. Removal of non-soluble contaminants was carried out by centrifugation at 22,000 g, 15° C. for 30 minutes. The supernatant was recovered, and collagen was precipitated by slowly adding crystalline NaCl to a final concentration of 3.13 M while constantly stirring at R.T. for 20 minutes. The solution was brought to O.N. Incubated. Collagen was collected by centrifugation at 25,000 g at 5° C. for 2 hours.

The supernatant was carefully poured through the 4 layers of a gauze pad. The pellet was resuspended in 200 ml of 250 mM acetic acid and 2 M NaCl for 5 minutes using a magnetic stirrer. The suspension was centrifuged at 5° C. at 25,000 g for 40 minutes. Traces of the supernatant were removed from the glass vial. The pellet was redissolved in 200 ml of 0.5 M acetic acid for 1 hour at room temperature. Removal of non-soluble material was carried out by centrifugation at 15° C. at 16,000 g for 30 minutes. The supernatant was poured through 12 layers of gauze pad. Collagen was precipitated by slowly adding NaCl to a final concentration of 3 M while constantly stirring at R.T. for 20 minutes. The solution was kept at 4° C. for 8 hours to O.N. Incubated for the following. Collagen was collected by centrifugation at 25,000 g at 5° C. for 2 hours. After aspiration of the supernatant, the pellet was redissolved in 200 ml of 0.5 M acetic acid using a magnetic stirrer for 1 hour at R.T. Removal of non-soluble material was carried out by centrifugation at 15° C. at 16,000 g for 30 minutes. The supernatant was poured through 12 layers of gauze pad. Collagen was precipitated by slowly adding NaCl to a final concentration of 3 M while constantly stirring at R.T. for 20 minutes. The solution was incubated at 4° C. for 8 hours. Collagen was collected by centrifugation for 2 hours at 2,000 g at 5°C. The supernatant was aspirated. The pellet was redissolved by pipetting in 40 ml of 10 mM HCl and vortexed for 5 min at R.T. The solution was transferred to a dialysis bag (MWCO 14,000 Da) and dialyzed at 4° C. against 4 L of 10 mM HCl for 4 hours. O.N. Repeated.

The solution was first filtered through a 0.45 μm filter using a 30 ml syringe, and then filtered through a 0.2 μM filter, thereby sterilizing collagen. Collagen was further concentrated through ultrafiltration using a Vivaspin PES 20 ml filter tube (Vivascience, #VS2041, MWCO 100'000). Centrifugation was carried out for 45 minutes at 5,000 g at 5° C. until the volume was reduced to 0.75 ml.

Optimization of the conditions of digestion kinetics and procollagen cleavage by food-grade picin: pellets until saturated (collected as described in Example 10) in 25% ammonium sulfate (AMS) buffered (buffer A: 0.1 M 4.36 g pellet in 4.5 mM potassium metadisulfite, 12.5 mM L-cysteine, 7.5 mM EDTA) dissolved in sodium phosphate buffer and titrated to pH 7.5 with 10 M NaOH or 6 N HCl: resuspended in a ratio of 200 mL ice-cold buffer Made it. After that, the sample was stirred at 15° C. for 20 minutes. Thereafter, aliquots of 10 mL were prepared per 15 mL test tube, followed by administration of increasing concentrations (5 mg/L to 15 mg/L) of ficin (pig tree latex, Biochem Europe food grade ficin). Samples were incubated at 15° C. for 1 to 3 hours, separated by SDS-PAGE, and then analyzed by Western blot for the presence of collagen migrating at a lower molecular weight than procollagen.

Tobacco leaf-derived pellets resuspended in phosphate buffer A (27.2 g: 800 mL buffer) at various pH values (5.5, 7.5, or 8.5) at 15° C. for 1 hour in the presence of 0 M to 3 M NaCl at 10 mg Treated with /L escape. The reaction was terminated by centrifuging a 1 mL sample from each reaction mixture (10 min, 15,000 g, 4°C). The pellet was resuspended in 1 mL buffer A (pH 7.5), separated by SDS-PAGE, and analyzed by Western blot.

Degradation kinetics and pharmacological-grade escape: pharmaceutical-grade (7.5, 8.5, 9.5) of various pH values with NaCl concentrations (0 M to 3 M) increasing conditions for procollagen cleavage by tobacco leaf pellets Biochem-Europe Pharm grade) resuspended in ficin-containing extraction buffer (10 mg/L) for 5 to 45 minutes. Further experiments studied the necessary and optimal conditions and concentrations of EDTA and L-cysteine as additives to the extraction buffer. Samples were incubated in the digestion mixture in the presence of 0 mM to 100 mM EDTA with 0 mM to 80 mM L-cysteine for 1 to 3 hours without NaCl at 15° C., pH 7.5.

Fibrilogenesis: Fibrilogenesis is considered as a collagen functional test. Therefore, the ability of purified collagen degraded by ficin to form fibrils is an essential property of the obtained product. Test method: The pH of the collagen-containing solution (two duplicate samples) was neutralized with sodium phosphate, pH 11.2 to pH 6.7, and then incubated at 27+/-2 μC for 6 hours. The sample was centrifuged to settle the formed hydrogel. The protein concentration of the sample before- and after-neutralization (supernatant) was determined by the Lowry method. PURECOL ^™ (NUTACON, Cat No. 5409) was used as a positive control and gelatin was used as a negative control.

Example 7. Extraction and purification of collagen from transgenic plants in the presence of trypsin and pepsin.

The production of human collagen in plants has been initiated to avoid the use of collagen from mammalian sources because the evolutionary proximity is relatively close and the use of mammalian proteins in human cosmetic or medical applications can be dangerous to human health. Known disease, Creutz-Jakob disease (CJD) is one of the diseases caused by consumption of infected mammalian proteins by humans.

Initially, purification of collagen from transgenic plants was performed using bovine pancreatic trypsin and digestive protease pepsin, both enzymes catalyzing the hydrolysis of proteins in the animal digestive system. The following examples illustrate the identification of proteases from non-animal sources suitable for use in a collagen purification process.

result

Propeptide digestion during the purification of collagen was first performed by the pancreatic enzyme trypsin. Trypsin degraded collagen propeptide at 300 mg/L, but the collagen yield was very low at the end of the purification process ( FIG. 10 ). When the concentration of trypsin was lowered to 20 mg/L or 30 mg/L, the yield was higher, but the procollagen degradation was only partial and was not consistent between the same samples ( FIG. 11 ).

To overcome this problem, various incubation temperatures and times were tried, but the results did not cause a change in yield (data not shown). In the purification process, the subsequent addition of pepsin enzyme solved the problem of partial degradation ( FIG. 12 ), yielding alpha-1 and alpha-2 collagen, and these collagens co-migrated together with the pig-derived collagen control sample.

Example 8. Collagen extraction and enzymatically-induced degradation thereof.

However, the trypsin-pepsin solution was not optimal because it required two different enzymes to prolong the purification process. Moreover, both enzymes are derived from animal sources. To overcome these problems, screens of different protease enzymes of non-animal origin were performed. Various digestion patterns were obtained by various enzymes screened. When collagen was incubated with the enzymes sabinase ( FIG. 15 ) and esperase ( FIG. 17 ), there was little or no observable degradation of the propeptide. Incubation with papain ( FIG. 14 ), bromelain ( FIG . 13 ), 2.4 L of Alcalase and 8.0 L of Esperase ( FIG. 18 ) resulted in over- or under-decomposition of the propeptide. Alcalase and Protamex enzymes ( FIG. 16 ) resulted in the desired degradation pattern and level (25 mg/L, 6 hours), where the alpha 1 and alpha 2 chains migrated similarly to the pig-derived collagen sample. . However, not all molecules have been completely degraded and may require a longer incubation period. Optimal results were obtained when procollagen and ficin (5 mg/L and 25 mg/L) ( FIG. 15 ) were incubated, at this time, the bands of the alpha 1 and alpha 2 chains co-migrated with the pig-derived collagen control sample. There was no visible overdecomposition at this time. Similar results were demonstrated at 5 mg/L of subtilisin for 3 hours ( FIG. 13 ) and 25 mg/L for neutrases for 6 hours ( FIG. 17 ).

Example 9. Extraction and purification of collagen from transgenic plants after degradation by subtilisin or ficin.

Collagen purification was performed from the leaves (13-361 or 13-6-52) of 450 gr of transgenic plants, followed by digestion of procollagen by ficin ( FIG. 19 ) or subtilisin ( FIG. 20 ). Collagen samples were analyzed by Western analysis at various stages during the purification process. Propeptide degradation by ficin and subtilisin resulted in the desired degree of processing of collagen 1 and collagen 2. Bands of lower molecular weight were observed on Western blots throughout the purification process, but these bands appeared in plant extracts before incubation with enzymes (lanes 3-4) and also in pig-derived collagen control samples (positive control) ( FIG. ).

Example 10. Scale-up extraction and purification of collagen from transgenic plants after digestion by picin.

In a 4 L reactor (ESCO model EL-3) 1 kg of transgenic tobacco leaves were pre-cooled in 2 L extraction buffer (100 mM sodium phosphate buffer pH 7.5, 4.5 mM potassium metadisulfite, 12.23 mM L-cysteine and 7.5) mM EDTA) for 20 minutes (5° C., 50% scraper speed and 100% homogenizer blade rpm). 6.68 g charcoal and 16.67 g PVPP were added to the extract and stirring was continued for 20 minutes (5° C. and 50% scraper speed). The extract was centrifuged (11,000 rpm, 5° C., 0.5 H), and the supernatant was saturated with 15% ammonium sulfate (1 hour stirring, 5° C.). After 6880 rpm, 5° C., 30 minutes, the supernatant was saturated to 25% ammonium sulfate, and stirred for 1 hour (5° C.). After recentrifugation, the pellet (6880 rpm, 5° C., 30 min) was resuspended (in extraction buffer) at 15% of the volume collected after the first centrifugation step. Removal of the propeptide was made possible by digestion for 3 hours at 15°C with 5 mg/L ficin (Biochem Europe). The sample was centrifuged (11,000 rpm, 15° C., 30 minutes), and mature collagen was precipitated using 3 M NaCl (NaCl was slowly added while stirring and left at 4° C.). After precipitation (13,000 rpm, 5° C., 2 hours), the supernatant was discarded and the pellet was resuspended in 0.5 M acetic acid. Another round of 3M salting out (O.N) and centrifugation was performed, followed by resuspending the pellet in 40 ml of 10 mM HCl. Samples were transferred to a dialysis bag (12 kDa to 14 kDa) and dialyzed at 4° C. against 4 L of 10 mM HCl for 4 hours. Dialysis was repeated with fresh 4 L 10 mM HCl, O.N. The dialyzed solution was filtered through a 0.45 micron filter (previously washed with 10 mM HCl), followed by a 0.25 micron filter. Finally, the samples were concentrated in a Vivaspin (Vivascience) filter tube (100 kDa).

Example 11. Solubility of Atelocollagen Produced as Recombinant Human Procollagen in Transgenic Tobacco Plants.

The concentration of atelocollagen produced according to Examples 9-10 was assayed by the following two methods as described in the Methods section. The concentrations obtained from some typical formulations degraded by picin are listed herein in Table 4 below:

Example 12. Ficin-dependent proteolysis of tobacco leaf-derived procollagen.

Degradation kinetics of procollagen by food-grade picin: To compensate for the appropriate picin concentration and incubation time to enable the highest collagen yield, increasing concentrations of food-grade picin (5-15 mg/L) at 15° C. for 1 to 3 hours. Thereafter, the samples were analyzed by immunodetection of alpha-1 and alpha-2 collagen chains on Western blot. Increased ficin concentration provided an improvement in collagen chains after a 1-hour incubation period ( FIG. 22 , lane 5 versus lane 6). However, upon prolonging the reaction time, the increased ficin concentration resulted in hyperdegradation of collagen ( FIG. 22 , lane 11 versus lanes 12-14 and lane 17 versus lane 18-20). Therefore, optimal conditions for the degradation of procollagen to collagen were set at 15° C. for 1 hour upon addition of 10 mg/L food-grade picin.

The kinetics of degradation of procollagen by pharmaceutical-grade picin: Similar experiments were conducted on procollagen-expressing tobacco leaf pellets to determine the conditions appropriate for procollagen degradation by pharmaceutical-grade picin. The pellets were resuspended and incubated for 0.5 to 3 hours at 15° C. with increasing concentrations of drug-grade picin (2.5 to 10 mg/L). Degradation efficiency was determined by immunodetection of collagen chains on Western blot. 23A-23C , increasing ficin concentration resulted in increased collagen production and decreased procollagen levels. The most effective degradation of procollagen by pharmaceutical-grade picin was observed at 10 mg/L after a 1-hour reaction time.

Optimization of pH value and salt concentration for ficin-dependent procollagen cleavage: Then, the contribution of both the digestion buffer pH and salt concentration was evaluated. Similar tobacco leaf post-AMS pellets were resuspended in extraction buffer titrated to pH 5.5, 7.5, 8.5 or 9.5 with salt contents ranging from 0.5 M to 3 M NaCl. The samples were then incubated with 10 mg/L drug-grade picin for 1 hour at 15° C. prior to immunoassay on Western blot. Acidic assay conditions (pH 5.5) resulted in insufficient collagen yield ( Figure 24A , lanes 2 to 6), while an increase in pH value demonstrated a correlated rise in picin-dependent collagen content, with a peak value of 2 It was observed at pH 8.5 in the presence of M NaCl ( FIG. 24B , lane 10). These results were further supported in scale-up extraction and purification experiments performed on two 15 kg pellets pooled for picin-induced procollagen digestion. Apart from the increased collagen chain yield as observed by immunoblotting, samples digested in a buffer at pH 8.5 in the presence of 2 M NaCl were efficiently raw as if digested in buffer A (pH 7.5, 0 mM NaCl). It was fibrous (see Table 5 below herein-batches YC1 and YC2). Therefore, both higher pH and salt concentration provided improved collagen yield after ficin-induced digestion of procollagen.

Determination of the vitalness of EDTA and L-cysteine in the digestion reaction mixture: Both EDTA and L-cysteine are additives present in the extraction buffer in the early stages of the collagen purification process. Here, the essentiality of these two components for effective escape-dependent collagen cleavage was determined. The procollagen post-AMS pellet was resuspended in extraction buffer containing increasing concentrations of EDTA (8 mM to 80 mM) and L-cysteine (10 mM to 100 mM), and with ficin (10 mg/L). Incubated at pH 7.5 and 15° C. for 1 hour. A pronounced enhancement effect was observed in the degradation efficiency in the presence of 10 mM L-cysteine ( FIG. 25 , lane 7 to lane 10), where there was no apparent contribution of EDTA to the ficin-dependent collagen result ( FIG. 8 to lane 10).

Optimization of temperature conditions for picin-induced procollagen degradation: Procollagen-expressing tobacco leaf pellets were incubated with picin at 15° C. for 1.5 hours and then transferred to a 30° C. bath for an additional 1.5 hours. Western blot and fibrilogenesis assays did not identify any improvement in collagen yield or sample purity with respect to increased reaction temperature.

Fibril formation of collagen extracted from ficin-induced cleavage of procollagen: The ultimate method of determining the ability of the produced collagen to form fibrils and the functionality of collagen by performing fibril formation assay after ficin-induced degradation Was determined. Table 5 below summarizes the fibril formation results as determined after ficin digestion of procollagen using two modified protocols. Both Protocol A and Protocol B with different reaction buffer pH and salt content yielded significant percentages of collagen fibrils. Therefore, the proteolytic reaction parameters developed and optimized herein resulted in high yield of functional collagen.

Example 13. TRYPZEAN in procollagen cleavage ^TM Determination of protease efficacy.

Procollagen -expressing tobacco leaf pellets resuspended in extraction buffer (pH 7.5) enriched with EDTA (7.5 mM) and L-cysteine (12.5 mM) were 1 at 15°C with TRYPZEAN ^™ ( 30-100 mg/L). Incubated for hours to 3 hours. After 1 hour, the 60 mg/L and 100 mg/L doses of TRYPZEAN ^TM efficiently cleaved procollagen, yielding two distinct alpha collagen chains, and there was no detectable hyperlysis ( FIG. 26 ). Therefore, treatment of procollagen by TRYPZEAN ^™ at pH 7.5 resulted in effective degradation into collagen chains alpha-1 and alpha-2.

Argument

Examples 7 to 13 above describe the identification of non-mammalian proteases suitable for use in the purification process of plant-derived collagen. Proteases from bacterial and plant sources were tested and three enzymes, neutrase , subtilisin, TRYPZEAN ^™ and ficin, were found to be suitable for collagen propeptide digestion.

Both neutrase and subtilisin are secreted by the bacterial Bacillus species. Subtilisin is mainly used (>90%) in detergents and household cleaning products. Approximately 10% of subtilisin uses are directed towards technology applications such as proteolysis, leather treatment, and the textile and beauty industries. At higher concentrations, the standard use of subtilisin in the collagen purification process is a problem due to overdegradation of collagen. Neutrases are mainly used in the beverage alcohol industry and in cheese aging. In Examples 7 to 13 described above herein, neutrases were only effective in degrading propeptide at high concentrations, and at least 6 hours were required for the desired decomposition results.

Under the experimental conditions currently described, recombinant trypsin and ficin were found to be the most stable of the four because there was no hyperlysis of collagen at high enzyme concentrations or after extended incubation periods. Moreover, these enzymes apparently did not degrade the helix regions of collagen as determined by SDS PAGE analysis. Pigment latex plants (P kusu car Rica (Ficus carica)) of escaped the natural enzyme extract for is commercially available at a low cost in several grades, including pharmaceutical grade from several sources. It is used in the food industry: alcohol and beer industry, hydrolisation of proteins, meat processing, confectionery industry, and pet food and health food manufacturing. It also applies to the pharmaceutical industry, contact lens cleaner, cancer treatment, anti-arthritis treatment, and digestive aids, as well as the cosmetic and textile industries.

Example 14. Further analysis of rh collagen properties.

Materials and methods

material

Human recombinant collagen (rh collagen) type I expressed and isolated from transgenic tobacco plants was generated and supplied by CollPlant Ltd (Israel). Type I bovine collagen (PureCol) was purchased from Advanced Biomatrix, USA. Methacrylic anhydride, glycidyl methacrylate, triethylamine, tetrabutylammonium bromide, 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2-hydroxy-4'-(2-hydroxyethoxy) -2-Methylpropiophenone (Irgacure 2959), sodium phosphate monobasic anhydrous, HCl 1 N, HCl ≥37%, sodium bicarbonate and NaOH were purchased from Sigma Aldrich Ltd, Israel. Phosphate buffered saline (PBS), x10 PBS, fetal bovine serum (FBS), DMEM high glucose and penicillin/streptomycin were purchased from Biological Industries Ltd, Israel. Sodium phosphate dibasic anhydride was purchased from Canton, India. Absolute ethanol and acetone were purchased from Bio-Lab Ltd, Israel. Hyaluronic acid was purchased from Lifecore, USA.

Preparation of buffer and photoinitiator stock solutions

Fibrilization Buffer (FB): Sodium phosphate dibasic was dissolved in double distilled water (DDW) to a final concentration of 162 mM. The solution was titrated to pH 11.2 using 10 N NaOH.

Medium preparation: 50 ml of fetal bovine serum and 5 ml of penicillin/streptomycin (10,000 units/mL and 10 mg/mL, respectively) were added to 500 ml of DMEM high glucose medium under sterile conditions. The medium was gently mixed and kept in a freezer.

Phosphate buffered saline preparation: 39 ml of 0.1 M sodium phosphate monobasic solution was mixed with 61 ml of 0.1 M sodium phosphate dibasic solution and the final volume was adjusted to 200 ml with DDW. If necessary, the final pH was adjusted to 7 with concentrated NaOH or HCl. NaCl was added to a final concentration of 150 mM.

Wash Buffer: HCl was added to the fibrillar buffer to reach a final concentration of 16.2 mM sodium phosphate dibasic and 10 mM HCl. The pH was adjusted to 7.2 to 7.4 with 10 N NaOH.

Photoinitiator 10% (v/v) stock solution: Irgacure 2959 was dissolved in absolute ethanol/PBS 1:1 solution to a final concentration of 100 mg/mL.

methacrylate of rh collagen

Fibrillar rh collagen-methacrylamide and monomeric rh collagen-methacrylamide were prepared by reacting lysine and hydroxylysine collagen residues with methacrylic anhydride in an aqueous medium as described below, until further use at 4° C. Stored under light protection.

Fibrillar rh collagen-methacrylamide

3-10 mg/mL fibrillar rhcollagen-methacrylamide was synthesized in wash buffer, fibril formation buffer or DDW, at room temperature (RT) or 12°C. For example, briefly, fibrillar collagen-MA was synthesized in DDW as follows: a solution of 3-4 mg/mL of monomeric rhcollagen in 10 Mm HCl (COLLAGE ^TM ) was mixed with fibril formation buffer and 9: Mixing at a ratio of 1 v/v and stirring at RT for 1 hour to obtain fibrils. The solution was centrifuged at 4° C. at 7500 rpm for 30 minutes, and the supernatant was discarded. The pellet was resuspended in the same volume of wash buffer and centrifuged under the same conditions. Thereafter, the sedimentary fibrils were resuspended in DDW to 10 mg/mL. The concentration was confirmed by solid percentage measurement. Methacrylic anhydride (MA) was added dropwise at a molar ratio of 10-20 to collagen lysine at room temperature under nitrogen flow, and the reaction solution pH was monitored over time and adjusted to pH 7 with 10 N NaOH. After 24 hours reaction, in wash buffer (pH 7) using 10 kDa cut-off dialysis tubing (Spectrum Laboratories Inc, CA, USA) for 3 days at 4° C. with at least 6 dialysate (in this case wash buffer) changes. The mixture was dialyzed against the reaction to remove reaction by-products, and finally lyophilized for 3 to 4 days.

Monomeric rh collagen-methacrylamide

200 mM MOPS, phosphate, or Tris buffer was used with the addition of 150 mM NaCl. For example, 200 mM MOPS and 150 mM NaCl were added to 3-4 mg/mL COLLAGE ^™ and stirred at RT until a clear solution was obtained. Then, 10-fold to 20-fold excess of methacrylic anhydride was added dropwise at 12° C. under nitrogen flow, and the pH was adjusted to pH 7 over time using 10 N NaOH. After 24 hours reaction, the mixture was dialyzed against 10 mM HCl and 20 mM NaCl (pH 2) in 10 kDa cut-off dialysis tubing for 3 days at 4° C. with at least 6 dialysate changes, followed by 3 days to It was lyophilized for 4 days.

Methacrylate of hyaluronic acid (HA)

500 mg of HA was added to Leach et al. [Leach et al. 2002, Biotechnology and Bioengineering, vol. 82, no. 5]. Briefly, 1.8 ml of triethylamine, 1.8 ml of glycidyl methacrylate, and 1.8 g of tetrabutyl ammonium bromide are separately added to 50 ml of a 10 mg/mL HA solution in DDW, and the following components are added. Mix thoroughly before doing. The reaction was mixed overnight at room temperature, and HAMA was precipitated in 20-fold volume of acetone and redissolved in DDW. The precipitation process was repeated twice to remove all reaction residues. The material was eventually lyophilized.

Solution preparation for viscosity measurement

By adding 1 ml of PBS X10, PureCol and Collage™ in PBS: 8 ml of monomeric collagen solution (3 mg/mL in 10 mM HCl), rh collagen (COLLAGE ^™ ) or bovine collagen (PureColl) were neutralized. Thereafter, the solution was brought to pH 7 to 7.5 by titration with 0.1 N NaOH. Finally, double distilled water was added to reach a final volume of 10 ml. After incubating the samples at 37° C. for at least 90 minutes, measurements were taken (at 37° C. or 4° C.).

COLLAGE™ in fibril-forming buffer: 9 ml of a solution of monomeric rh-collagen (COLLAGE ^™ ) (3.79 mg/mL in 10 mM HCl) in fibril-forming buffer was neutralized by adding 1 ml of fibril-forming buffer. After incubating the samples at 37° C. for at least 90 minutes, measurements were taken (at 37° C. or 4° C.).

Fibrillar rhcollagen-methacrylamide in PBS: 10 mg in PBS of lyophilized fibrillar rhcollagen-MA (with 10-fold excess MA as described above) prepared in DDW and dialyzed against wash buffer It was dissolved to a concentration of /mL. After incubating the samples at 37° C. for at least 90 minutes, measurements were taken (at 37° C. or 4° C.).

Rhcollagen-methacrylamide in DMEM: lyophilized fibrillar rhcollagen-methacrylamide (15-fold excess of MA prepared and dialyzed in wash buffer as described above) in DMEM medium at 20 mg/mL and 26 It was dissolved to a final concentration of mg/mL.

Rhcollagen-methacrylamide/hyaluronic acid in DMEM: hyaluronic acid was added to a solution of fibrotic rhcollagen-MA to obtain final concentrations of 10 mg/mL HA and 20 mg/mL rh collagen-MA in DMEM medium I did.

Rh collagen-methacrylamide/hyaluronic acid methacrylate (HA-MA) in DMEM: hyaluronic acid methacrylate (see above) was added to a solution of fibrotic rh collagen-MA, and 10 mg/mL in DMEM medium Final concentrations of HA-MA and 20 mg/mL rhcollagen-MA were obtained.

Rh-collagen-MA photocrosslinking for measurement of loss factor and storage factor

The rhcollagen-MA crosslinked scaffold was formed in two different formulations and was aimed to be tested in two separate experiments. In the first formulation, 1% to 2% by weight of fibrillar rhcollagen-MA synthesized with a 10-fold excess of methacrylic reagent was dissolved in 0.1 M PBS at RT, then 0.1% of Irgacure 2959 was added. , 1 mL final volume of the solution was injected into a discoid mold. Thereafter, a curing process was performed from a distance of 1.5 cm for 7 seconds and 10 seconds at an averaged intensity of 670 mW/cm ² using a mercury light source, and the curing process was terminated on a crosslinked scaffold. The second formulation contained two different batches of fibrillar rhcollagen-MA, synthesized with a 15-fold and a 20-fold excess of methacrylic reagent. 1% to 2% by weight was dissolved in 0.1 M of PBS and 0.1% of Irgacure 2959 was added to achieve a final volume of 1.5 mL. In order to obtain a highly crosslinked scaffold, a curing process was performed from a distance of 2 cm for 60 seconds at an averaged strength of 420 mW/cm ² .

TNBS assay

The assay protocol is similar to that reported by Sashidhar et al., Sashidhar R.B., Capoor, A.K., Ramana, D, Journal of Immunological Methods. 1994, 167, 121-127, and Habeeb [Habeeb A.F.S.A, Analytical Biochemistry. 1966, 14, 328-336]. Briefly, 0.4 mL of just prepared 0.01% (v/v) TNBS was added to 0.4 mL of 0.1-2 mg/mL fibrillar rhcollagen-MA in 4% sodium bicarbonate. After 2 hours reaction at 40°C, 0.2 mL of 1 N HCl and 0.4 mL of 10% (v/v) SDS were added. The absorbance was measured at 335 nm in a spectrophotometer in a 1 mL polystyrene cuvette. A control (blank) was prepared using the same procedure, except that sodium bicarbonate buffer was added instead of the rhcollagen-MA solution. The absorbance of 1-2 mg/mL native fibrotic rh-collagen prepared under the same conditions was recorded for calibration.

Rheological characterization

Viscosity: Viscosity measurements were performed using a C60/1°Ti cone-plate setup on a HAAKE RHEOSTRESS600 ^™ rheometer (Thermo Electron Corporation) equipped with a temperature-controlled cell chamber. Viscosity was measured on 1 mL samples at 4° C., 25° C. and 37° C. in a rotary ramp mode with shear rates ranging from 0.0001 to 1000 sec-1.

Scaffold storage factor and loss factor: A parallel plate system using a PP20 serrated spindle and a 20 mm serrated plate setup was used to investigate the rheological behavior of rh collagen crosslinked discs. To characterize the non-crosslinked rhcollagen-MA, a C60/1°Ti cone-plate element was used. To evaluate the rheological behavior of rhcollagen-MA, two sets of experiments were performed individually. In the first experiment, a 1 mL sample was subjected to an oscillation force in a controlled stress mode, applying a 5 Pa shear stress at a frequency of 1 Hz and 37°C for 300 seconds while the storage factor G'and the loss factor G' 'Was recorded. The gap was adjusted to 90% of the original sample, and the G'and G'' values were averaged in the range of 150 seconds to 300 seconds. In the second experiment, a 1.5 mL crosslinked disc was tested in frequency sweep vibration at 37°C, where G'was recorded under 1 Pa shear stress in the frequency range of 0.01-100 Hz. To initiate the measurement, the spindle was lowered and brought into contact with the hydrogel surface, after which it was further lowered until the axial force of the instrument was 0.4 N. Prior to all measurements, the sample was held for 1 minute on a plate covered with a humidity stopper to reach temperature equilibrium.

result

TNBS assay

The degree of modification of rh collagen was quantified using the TNBS colorimetric assay. This assay quantified the molar content of free, unreacted -amino groups derived from lysine and hydroxyl lysine, and subsequently the degree of functionalization. The degree of functionalization of the fibrillar rhcollagen 10-fold, 15-fold and 20-fold different batches was determined by the TNBS assay as shown in Table 6.

The results suggest that the fibrillar rh-collagen has a high modification ability, and that the addition of a methacrylic reagent at a molar ratio of 10 may be desirable to receive maximum functionalization of fibrillar collagen.

Rheology

1. Viscosity

Temperature dependence of rh collagen/bovine collagen viscosity

Figure 27 shows the viscosity of rh collagen (COLLAGE™) and bovine collagen (PureCol) in PBS expressed as a function of shear rate at T=4°C (blue, dashed and solid lines, respectively) and T=37°C (red, respectively, Dashed and solid lines). Bovine collagen (solid line) is a zero-shear rate viscosity (η0) with a value of 37°C (red) η0 that is more than one digit higher than the value at 4°C (blue), i.e., the apparent temperature of the viscosity flat area at low shear rate values Show dependence. In contrast, rh-collagen (dashed line) shows no significant difference between the η0 value at 4°C and the η0 value at 37°C. Neutralized rh collagen in FB (see method) shows very similar behavior ( FIG. 28 ), ie the viscosity at 4° C. and 37° C. is almost the same. Figure 29 shows the viscosity of fibrillar rhcollagen-MA at 4°C (blue line) and 37°C (red line). Although the profiles are not identical, the zero shear rate value is about 1000 cP at both temperatures.

viscosity of rh collagen-methacrylamide

FIG. 30 shows the viscosity of rh collagen-MA dissolved in DMEM at 25°C. The typical shear thinning behavior of rh collagen shown in Figures 27 and 28 is also maintained for rh collagen-MA with and without HA/HA-MA. Increasing the concentration of rh-collagen from 20 mg/mL to 26 mg/mL (green line and red line, respectively) increases the zero-shear viscosity as well as by the addition of 10 mg/mL HA or HAMA. Resulting in a final polymer concentration of 30 mg/mL.

Those of skill in the art will recognize that rhcollagen-MA is not crosslinked and that in order to achieve crosslinking the person skilled in the art must add a photoinitiator and light.

2. Scaffold loss factor and storage factor

G′). G″, "손실/점성 계수"는 내부 마찰을 통한 전단 변형 시 상실되는 G*의 에너지 분율을 나타낸다. HA 충전제가 순수하게 점성이 아니므로 G"는 점도와 직접적으로 관련이 있지 않다. 대신에, 이 용어는, 전단 응력이 제거된 후 겔이 이의 형상을 완전히 회복시키지 못하는 무능력을 반영한다. Fig. 31 shows the rheological analysis of a 1 mL disc over time at 37°C performed in the first experiment. The upper graph reports the loss factor, storage factor and tan (delta) before UV curing, while the lower graph reports the above values after UV curing (when photoinitiator is added). The data demonstrates that the storage coefficient of rhcollagen-MA increases by 2-fold upon illumination in the presence of a photoinitiator. Moreover, the results indicate the ability to modulate scaffold properties by varying the rhcollagen-MA concentration. The high difference between the G'and G'' values of the crosslinked disk, and the near-zero tan (delta) value, indicate its elastic-like behavior. (G'-storage modulus; G''-loss factor; G', "storage/elastic modulus" represents the energy fraction of G* that is stored by the gel during deformation and subsequently used to restore its original shape. G'measures the elastic behavior of a gel, or how much the gel can recover its shape after shear deformation, for example, a vulcanized rubber deforms immediately under stress and fully recovers its shape after the stress is removed. Therefore, it is a purely elastic material (i.e. G*

G′). G″, “Loss/Viscosity Coefficient” represents the energy fraction of G* that is lost during shear deformation through internal friction. Since the HA filler is not purely viscous, G" is not directly related to viscosity. Instead, the term reflects the inability of the gel to fully recover its shape after the shear stress has been removed.

In the second experiment, a 1.5 mL disc irradiated for 60 seconds gave a higher G'value as shown in FIG . 32 . The data show that G'increases with rhcollagen-MA concentration and degree of methacrylation, indicating the ability to modulate scaffold properties.

Example 15. Procedure for obtaining and processing rh collagen from tobacco plants.

Genetically modified tobacco plants are grown as described above, and leaves are harvested and prepared for initial upstream extraction and purification ( FIGS. 33A-33C ). As shown in Figure 33A , the leaves are subjected to mechanical shredding ( step A ), the pulp is removed from the slurry, while the procollagen-containing moiety is retained and subjected to enzymatic digestion to convert the procollagen to collagen. Conversion ( Step B to Step C ). The pulp is discarded again, and the collagen-containing moiety is retained from the sludge ( step C ). After the acidification step, the sample undergoes a first centrifugation during the first washing step, after which the pellet is discarded ( step DF ). After AMS precipitation and second centrifugation, the protein is precipitated (H pellet) and the supernatant is discarded ( step GH ). H pellets can be frozen at -20°C for storage.

As shown in FIG . 33B , the H pellet is resuspended to yield a protein suspension, followed by a third centrifugation, after which the pellet is discarded ( step IJ ). The suspension is washed using a depth filter, and the suspension is salted out with NaCl to precipitate collagen ( step KL ). The fourth centrifugation yields a collagen pellet and discards the supernatant ( step M ).

As shown in Figure 33C , the collagen pellet is resuspended in HCl to yield solubilized collagen ( step N ). After 0.2-0.8 micron filtration, ultrafiltration (UF) (concentrated and diluted filtration) results in bulk concentrated collagen ( step OP ). After 0.2 micron filtration and filling, the purified collagen is stored in its final container ( step Z ).

Example 16. Viscosity and polymerization of rh collagen methacrylate and additives.

Different additives (polyvinyl alcohol methacrylate (PVAMA) ( FIGS. 34 and 37 ), hyaluronic acid methacrylate (HAMA) ( FIGS. 35 and 37 ) in the ratio of collagen MA: additive of 5:1, 2:1 and 1:2 37 ), and 5 mg/ml rh collagen methacrylate concentrated with oxidized cellulose (OC) ( FIGS. 36 and 37 ) are shown in FIGS. 31 to 37. Viscosity of 5 mg/ml rh collagen methacrylate is compared (Black curve) All samples were prepared in 0.1 M phosphate buffer pH 7.4 + 11.3 mM NaCl (physiological osmolarity) and measurements were carried out at T=22° C. Data comparison And summarized in FIG. 37 .

Polymerization of rh collagen methacrylate enriched with different additives is also shown with respect to a typical scaffold of 5 mg/ml collagen MA+ different additives at a ratio of collMA: additive 2: 1 ( FIG. 38 ). ColMA alone was compared to ColMA in combination with polyvinyl alcohol methacrylate (PVMA), hyaluronic acid methacrylate (HAMA), or oxidized cellulose (OC). The solution was mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.1%), and irradiated with ultraviolet (uv) light (365 nm) for 20 seconds.

Example 17. Injectable rh collagen/platelet rich plasma scaffold.

The rh collagen/platelet rich plasma (PRP) scaffold for injection was investigated as a scaffold for tendonopathy and a means of cure. A source of growth factor (GF), such as a slow-degrading rh collagen matrix in combination with platelet rich plasma (PRP), was injected near the injured tendon to provide the support necessary to enhance healing of the injured tendon. . The treatment used a matrix made of plant-derived recombinant human type I collagen (rh collagen) mixed with PRP, which supports prolonged release of growth factors at the injured site and promotes healing. Comparison of the effect of rh collagen-PRP matrix with PRP in vitro and in vivo in supporting fibroblast proliferation, thrombolysis, release of GF and tendon healing in a collagenase-induced Achilles tendon tendonosis rat model. I did. rh collagen-PRP demonstrated superior performance in vitro and in vivo compared to PRP alone. These results are encouraging regarding the use of rh collagen matrix in combination with PRP in clinical trials for tendonopathy indications.

Materials and methods

rh collagen matrix

A monomeric solution of rh collagen (CollPlant, Neschiona, Israel) in 10 mM HCl was fibrilized by pH neutralization in a phosphate solution, and 18 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ( Sigma Aldrich, Israel). Thereafter, the crosslinked collagen was washed by repeated centrifugation in double distilled water, calcium chloride (CaCl2) (Merck, Israel) was added, and the final concentration of 20 mM was calculated. The syringe filled with rh collagen slurry was lyophilized and finally sterilized with ethylene oxide.

Platelet rich plasma (PRP) production

Granulocyte-free PRP was prepared using the Tropocell PRP kit (ESTAR, Israel) according to the manufacturer's instructions. For the in vitro cell proliferation assay, human blood was drawn from healthy human volunteers (Helsinki License No. 2012068). For in vivo animal studies, blood was drawn from Hsd:Sprague DawleySD rats (Harlan).

Rh collagen matrix / PRP and control preparation

Rh collagen matrix/PRP: A syringe containing lyophilized cross-linked rh collagen was hydrated with PRP or saline to obtain a final concentration of 20 mg/ml rh collagen.

Thrombin Activated PRP (Control): Human PRP was mixed with purified thrombin (Sigma Aldrich, Israel) to obtain a final concentration of 100 IU/ml.

CaCl2 activated PRP (control): Rat PRP was mixed with CaCl2 (Merck, Israel) to obtain a final concentration of 20 Mm.

In vitro cell proliferation assay

In this study, the effect of GF on normal human dermal fibroblast (nHDF) viability and proliferation was evaluated. Cell viability and proliferation were compared upon GF diffusion from a matrix consisting of a crosslinked rh collagen matrix combined with PRP or from a thrombus consisting of thrombin activated PRP. Rh collagen matrix in combination with PRP or thrombin activated PRP (200 μl each) was placed on top of a 24-well plate (Thermo scientific, Israel) transwell (Thincerts™ 24-well 8.0 μm, Greiner bio-on, Israel) ) And incubated at 37° C. for 20 minutes to form a thrombus. Normal human dermal fibroblasts (nHDF) (5,000 cells per 0.5 ml) were inoculated at the bottom of each well in serum depleted medium (Dulbecco's Modified Eagle's Medium, DMEM, 1% fetal bovine serum, FBS, Biological Industries, Israel). I did. Transwells containing a matrix (PRP or rh collagen matrix in combination with thrombin activated PRP) were placed on top of the inoculated wells, and an additional 0.2 ml of medium was added on top of the sample. NHDF in 0.5 ml DMEM, 1% FBS was inoculated as a control. After inoculation using the cell proliferation kit WST-1 (Roche, Israel) according to the manufacturer's instructions, the samples were tested in duplicate on the 7th and 10th days.

In vivo research

animal

Hsd:Sprague Dawley SD rats weighing 230 g±20% were selected for animal experiments. Animals were assigned unique animal identification ear numbers and randomly assigned to specific groups. Animals were housed in individually ventilated (IVC) cages in dedicated heat, ventilation, air conditioning (HVAC) animal facilities. Temperature and humidity were continuously monitored. Animals were provided with commercial rodent feed (Harlan Teklad TRM Ra/mouse feed) at will and had free access to autoclaved water. The facility was not exposed to external light and maintained in an automatic alternating cycle of 12 hours light and 12 hours dark. All animals were handled according to the laboratory animal treatment and care guidelines, and all protocols were approved by the local institutional animal care and use committee. No abnormalities were detected in any animals over the entire study period. No statistically significant differences were found in mean group weight values and acquisitions. All acquisitions were within the range of normally expected values at the end.

Breakdown of blood clots and release of growth factors in vivo

The degradation time and GF content of rh collagen matrix in combination with PRP, rh collagen matrix alone or CaCl ₂ activated PRP over time were compared in a subcutaneous (SC) rat model (Science in Action Ltd., Neschiona, Israel). I did.

The injection site on the back of 34 Sprague Dawley rats (Harlan Laboratories, Neschiona, Israel) was shaved and marked. In each rat, 0.5 ml of the same formulation was injected into four spaced positions on the rat's back, on the dorsal plane, at two sites in the anterior portion, and at two sites in the posterior portion. Animals were euthanized at post-treatment time points 1, 7, 14, 21, 30 and 45 days (10 or 12 animals per group, 2 animals per time point). At each time point, the injection site was exposed and evaluated macroscopically. The skin at the injection site was gently separated from the muscle using a scissor, and the site was washed with 0.25 ml DMEM, 1% FBS (Biological Industries, Israel), and the thrombi were extracted and weighed. Wash medium was transferred to Eppendorf tubes (1.5 ml to 2 ml), while extracted thrombi were transferred to 6 or 12 well plates. Once weighed, the thrombus was combined with each wash medium, cut with a scissor and minced with a pestle to promote the release of GF from the thrombus to the surrounding medium. Thereafter, the Eppendorf tube was centrifuged for at least 5 minutes to separate the blood clot pellet and the medium. The supernatant was collected and stored at -80 °C until assayed. Controls (T0) containing about 0.5 ml of each formulation were formed in vitro following the same procedure as described above without injection to animals. At the end of the study, the content of PDGF and VEGF in the preserved supernatant was evaluated by ELISA (Quantikine ELISA mouse/rat PDGF and Quantikine ELISA rat VEGF, R&D Systems, Israel).

In vivo tendonopathy induced in rats

In a collagenase-induced tendonosis model in 36 male Sprague Dawley rats (18 rats per group, 6 animals per time point), the healing properties of rh collagen matrix combined with PRP and PRP alone were compared. Experiments were carried out at Harlan Laboratories Israel Ltd. (Neschiona, Israel).

Skin incisions were made over the proximal of the rat's right hind limb over the common heel tendon. Under appropriate magnification, the central branch of the tendon was identified and isolated, and tendonopathy by injecting 0.3 mg collagenase (10 mg/ml, Sigma) under the common heel tendon sheath using a 0.5 ml insulin syringe. Was induced. Eventually, the skin was closed with an interrupted subcutaneous suture using 4/0 Vicryl. One week after tenopathy induction, a stab incision was made in the tendon sheath using an ophthalmic corneal/scleral knife. Thereafter, a tunnel was created under the tendon using a cannula, and rh collagen or PRP alone in combination with 50 μl of PRP was injected into the pre-generated canal. Animals were euthanized on days 3, 7 and 14 post-treatment. Treated tendons were excised and preserved for histopathological evaluation.

histology

The tissue was embedded in paraffin and successively cut into samples of 4 to 5 microns thick. For histopathological examination, slides were stained with hematoxylin and eosin (H&E) and evaluated blindly by a pathologist.

result

In vitro cell proliferation assay

In this study, the viability and proliferation of cells inoculated around thrombus consisting of a matrix consisting of rh collagen combined with PRP and thrombin activated PRP were compared. Cells inoculated into untreated wells were used as controls. A matrix (consisting of rh collagen or thrombin-activated PRP in combination with PRP) was placed on top of the inoculated wells in transwells to allow diffusion of GF from the matrix to the wells without direct contact with the cell layer. The number of living cells on days 7 and 10 is reported as the average of two different experiments (3 replicates per experiment) with PRP extracted from two different blood donors ( FIG. 39 top ). As shown in Figure 39 , cell viability (day 7 and 10) in the presence of GF released from the rh collagen matrix combined with PRP is significantly higher than in thrombin activated PRP thrombi or than in the control. Moreover, the number of cells increased from day 7 to day 10 in the presence of the rh collagen matrix in combination with PRP, while the number of cells in the presence of thrombin-activated PRP and in the control decreased, which indicates that both cell viability and proliferation are rh collagenase. It shows quite good in the presence of the matrix. The data was confirmed by microscopic analysis ( bottom of FIG. 39 ). Cells cultured in the presence of rh collagen matrix combined with PRP ( FIG. 39, bottom, panel A ) showed an elongated shape 7 days after inoculation and had already reached full abundance, while thrombin-activated PRP Few cells cultured in the presence were alive, which could indicate the toxic effect of thrombin in this experimental setup. ( Figure 39 bottom, panel B ). Cells cultured only in the presence of medium showed very limited viability ( Figure 39 bottom, panel C ).

In vivo matrix degradation profile and growth factor release

Matrix decomposition profile

The disintegration profile of the injected formulation was determined by weighing the matrix at different time points after subcutaneous injection into the rat.

Upon injection of activated PRP, the substance had already disappeared on day 1 ( FIG. 40 ), suggesting complete degradation of fibrin thrombi during the first 24 hours. On the other hand, the rh collagen matrix alone or in combination with PRP had a two-phase degradation profile ( FIG. 40 ), which profile started with a steep weight loss during the first day and followed by a relatively slower degradation rate. Complete removal was caused after days to 45 days (final weight was less than 0.5% of the initial weight).

Growth factor content

The GF content at the injection site as a function of time was assessed by ELISA for PDGF and VEGF ( FIGS. 41A to 41B ). The PDGF content at 0 hours was similar in rh collagen matrix combined with PRP and in activated PRP treatment ( FIG. 41A ), suggesting that the PDGF content on day 0 was the sole contribution of GF rich platelets made by PRP. However, upon injection of PRP alone, the PDGF content at the injection site was already lower than the detection limit on the first day after injection, and remained undetectable according to the entire study, consistent with rapid thrombus degradation ( FIG . Different pictures are shown when PRP is incorporated into the rh collagen matrix ( FIG. 41A ). The PDGF content gradually increased from day 1 to day 14, and decreased again until completely removed to day 45 consistent with scaffold degradation ( FIG. 40 ). Interestingly, the PDGF content in the rhcollagen matrix alone group increased starting from day 7, followed by the pattern presented by the matrix group combined with PRP. At day 0 the VEGF content was lower than the detection limit for all formulations and remained at baseline levels in the activated PRP group ( FIGS. 41A-41B and FIG. 42 ). The VEGF profile of the rhcollagen matrix in combination with PRP shows a rapid increase in VEGF content around day 7 followed by a steep decline by day 14 and flatness by day 30, VEGF eventually consistent with scaffold degradation. It drops on the 45th day ( FIG. 41B ).

The increase in GF seen from Days 1 to 14 in the PDGF assay and from Days 0 to 7 in the VEGF assay demonstrates the ability of the rh collagen scaffold to enable GF accumulation, migrating and proliferating in the scaffold. Tends to reflect cells. The integral of the nominal content of PDGF and VEGF over the entire study for each formulation is summarized in Figure 42 . It is clear that the GF content at the injection site is much higher upon injection of rh collagen matrix alone or of rh collagen matrix in combination with PRP compared to activated PRP alone.

In vivo tendonopathy induced in rats

The healing properties of rh collagen matrix in combination with PRP compared to PRP alone were evaluated in a rat model for tendonopathy, and at different time points by histopathological analysis. Tendon healing and inflammation were quantified by scoring the level of mature fibrosis, the presence of mononuclear inflammatory cells and the presence of immature granulated tissue (reserved 0-5 as described in Table 7).

The cumulative values of histopathological scores associated with each treatment are shown in FIGS. 43A-43C . The group treated with rhcollagen matrix in combination with PRP showed slightly more mature fibrosis, specifically on days 3 and 14 compared to the PRP treatment group. This was consistent and correlated with the lower levels of immature granulation seen by the group treated with rh collagen matrix combined with PRP on day 14 ( FIG. 43C ). Moreover, in FIG. 43B , the group treated with rh collagen matrix in combination with PRP exhibits a decrease in inflammation as indicated by the low presence of mononuclear inflammatory cells at all time points, especially on days 3 and 14. Overall, the data show that treatment of injured tendons with rh collagen matrix in combination with PRP is accompanied by a major decline in inflammatory mononuclear cells when compared to standard PRP injection treatment with higher levels of mature fibrosis and lower levels. Promotes faster healing as suggested by the immature granulation of.

Argument

Damage to soft tissues, including injuries to tendons and ligaments, is very common and causes a significant clinical burden. Although several treatments are available, their clinical benefit is still limited. This has encouraged the search for new alternatives with the intention of improving healing and shortening recovery time. Once mixed with PRP, it slowly degrades, induces cell migration and proliferation into the collagen scaffold, and enables prolonged release of GF at the injured area, forming a collagen-fibrin-PRP complex that better supports the healing process. Thus, an injectable matrix composed of human recombinant type I collagen was developed. In vitro experiments ( FIG. 39 ) showed significantly superior nHDF viability and proliferation in the periphery of the rh collagen matrix combined with PRP compared to thrombin activated PRP. The results demonstrated that sustained growth factors released from the collagen matrix promote and enhance cell proliferation. When combined with PRP, type I rhcollagen still provides a supportive environment that promotes and enhances cell proliferation even when not in direct contact with the cell layer. SC injection in rats initially showed that the GF-containing fibrin-thrombosis formed in in-situ during PRP injection was already degraded within 24 hours, and as a result, the GF content at the injection site was lower than the ELISA detection limit ( FIGS. 41A to Figure 41b ). Once platelets are complexed with the rhcollagen matrix, GF is released over 45 days, which is consistent with the scaffold degradation time ( FIGS. 40 and 41A-41B ). It is interesting to note that the GF content profile would have been expected by the standard release profile and thus is not monotonic. The PDGF profile in the PRP-treated rh collagen matrix ( FIGS. 41A to 41B ) demonstrated a first steep decline on day 1 very similar to the case of PRP alone, followed by a gradual increase until day 21, and scaffold. Consistent with the decomposition, a final decline to reach a non-detectable level was demonstrated. Interestingly, rhcollagen alone showed a gradual increase in a similar pattern of PDGF content during the first 2 weeks, and a decrease towards complete degradation of the scaffold. Thus, rhcollagen in conjunction with the PRP scaffold provides the benefits provided by the trapped PRP (initial GF release) and the benefits provided by the rhcollagen scaffold itself, which highly promotes and enhances cell recruitment and proliferation. Combine. Regarding the VEGF content profile, VEGF levels upon PRP injection remained very low throughout the entire study, while injection of rhcollagen matrix in combination with PRP resulted in an increase in VEGF levels within the first week, eventually leading to an increase in VEGF levels consistent with scaffold degradation. Caused. Interestingly, in contrast to PDGF, VEGF levels in the group treated with rhcollagen matrix alone were still higher than in the PRP alone group, but showed a different profile than the rhcollagen matrix in combination with PRP, especially on day 7. This observation highlights the contribution of PRP to GF levels once combined with the collagen scaffold. The healing properties of rh collagen and PRP were eventually compared with PRP in a rat model for Common Calcaneal tendon (Achilles tendon) tendonosis. Histological evaluation confirmed the ability of the rhcollagen matrix in combination with PRP to more rapidly establish mature fibrotic tissue, which is consistent with the scaffold ability to promote cell recruitment and proliferation as expected in previous experiments. Moreover, histological analysis also demonstrates that once the injured site is treated with rhcollagen matrix in combination with PRP, inflammation is substantially reduced compared to standard treatment with PRP alone.

This study demonstrates the biological effects in vitro and in vivo of an injectable scaffold consisting of crosslinked human recombinant type I collagen. Once combined with an autologous source of GF such as PRP, the formed scaffolds accelerate healing of soft tissue injuries by regulating the inflammatory response and promoting the faster formation of new healing tissue. The results suggest that the enhanced healing properties are in the unique combination of rh collagen and autologous PRP, which prolongs the release of GF. The data support the use of rh collagen matrix in combination with PRP in clinical trials for tendonopathy indications.

Example 18. Use of plant-derived human recombinant collagen as a dermal filler.

To reduce immunogenicity compared to tissue-derived human and bovine collagen, promote tissue regeneration, and provide a more homogeneous and potentially longer lasting dermal filler with improved rheological properties, human transgenic collagen (rh collagen) Is produced from plants (eg, genetically engineered tobacco plants), isolated, and used as a dermal filler. Typically, the genetically modified plant comprises an expressive sequence of at least one gene sequence of human deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-alpha, P4H-beta, and LH3. . Typically, the plant-derived human collagen comprises at least one modified human collagen alpha-1 chain as shown in SEQ ID NO: 3 and expressed in a genetically modified plant; And at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO: 6 and expressed in a genetically modified plant; The genetically modified plants further express exogenous prolyl-4-hydroxylase (P4H) (eg, human or other mammalian P4H). Optionally, the genetically modified plant expresses an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. For example, the human collagen alpha-1 chain is encoded by a sequence as shown in SEQ ID NO: 1 and/or the human collagen alpha-2 chain is encoded by a sequence as shown in SEQ ID NO: 2. Optionally, human collagen alpha-1 and/or alpha-2 chains are targeted to the vacuoles of plants or genetically modified plants, which are degraded to ficin, resulting in human atelocollagen.

Optionally, rhcollagen is formulated with or modified with other ingredients, including those known in the art for dermal fillers. Examples of modifications include, but are not limited to, methacrylation and/or thiolation. Examples of other ingredients include hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modification thereof. Derivatives, or any combination of these. Examples of other ingredients are hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP) , Calcium hydroxylapatite (CaHA), carboxymethylcellulose, crystalline nanocellulose (CNC), or combinations thereof. Modified derivatives of HA, PVA, PEG, or OC include, but are not limited to, photopolymerizable derivatives. Modifications of HA, PVA, PEG, or OC include, but are not limited to, methacrylate and/or thiolation. Examples of other components include, but are not limited to, initiators such as polymerizers or photoinitiators (eg, sensitive to visible, ultraviolet (uv) or infrared light). Examples of visible light photoinitiators include, but are not limited to, eosin Y+ triethanolamine or riboflavin. Examples of ultraviolet photoinitiators are lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2- Methylpropan-1-one (IRGACURE® 2959), including but not limited to.

An inherent property of tissue-extracted collagen is gelation at room temperature. At relatively low concentrations (eg, 5 mg/ml to 15 mg/ml) in physiological buffer, tissue-extracted collagen forms a gel upon transformation at low temperature (approximately 4° C.) to room temperature.

In contrast, rh-collagen has a relatively low viscosity (at the same concentration and formulation), so injection through a narrow gauge needle or cannula (27-gauge to 33-gauge) with a relatively lowered expression force, as well as It enables better transmission into smaller spaces, and greater flexibility in post-scan adjustment (shaping).

The rh collagen is placed in a syringe with a micro-gauge needle or cannula (27-gauge to 33-gauge) and injected into the void or space below the dermis. Thereafter, the injected rh-collagen is shaped, shaped or otherwise manipulated into the desired position (eg, via manual massage or using a shaping or shaping instrument such as a surgical depressor). Before, during or after this process, polymerization can be initiated by exposure to a light source (e.g., a light-emitting diode (LED), laser, or xenon lamp) located on or above the dermis that covers the injected formulation.

Example 19. Use of modified plant-derived human recombinant collagen formulated with photoinitiators and additives.

The rh collagen is modified by methacrylation as described in Example 18 . The modified rh collagen is prepared as a polymerizable solution formulation with a photoinitiator (eg Eosin Y+ triethanolamine or riboflavin). Hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, crystalline nanocellulose (CNC), or some combination thereof.

The formulation is placed in a syringe with a micro-gauge needle or cannula (27-gauge to 33-gauge) and injected into the void or space below the dermis. Then, as described in Example 18 , during or after exposure to a light source (e.g., a visible light source), manually or using suitable surgical instruments to shape, shape or otherwise shape the injected formulation into the desired position. If not, operate it.

Example 20. Use of modified plant-derived human recombinant collagen formulated with photoinitiators and modified additives.

The rh collagen is modified by methacrylation or thiolation as in Example 19 . The modified rhcollagen is prepared as a polymerizable solution formulation with a photoinitiator (eg Eosin Y+ triethanolamine or riboflavin) as described in Example 18 . Including modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or some combination thereof, and modified by methacrylate or thiolation Let it.

The formulation is placed in a syringe with a micro-gauge needle or cannula (27-gauge to 33-gauge) and injected into the void or space below the dermis. Then, as described in Example 18 , during or after exposure to a light source (e.g., a visible light source), manually or using suitable surgical instruments to shape, shape or otherwise shape the injected formulation into the desired position. If not, operate it.

Example 21. Comparative injectability and viscosity of crosslinked hyaluronic acid and collagen.

As shown in Fig . 44 , the expression power required for injecting crosslinked hyaluronic acid (HA) (black □ curve) expression power (Newton, N) was determined by monomeric collagen (▼ curve) or fibrilized collagen (▲ curve). ) With crosslinked hyaluronic acid (HA) was compared with the expression required for injection. (Crosslinked HA 20 ml/ml; crosslinked HA 20 mg/ml, monomeric rhCol 7.5 mg/ml; crosslinked HA 20 mg/ml, fibrilized collagen 10 mg/ml)

As shown in Figure 45, the expression force required to inject crosslinked HA (Newton, N) was compared with the force required to inject the formulation of double crosslinked HA-collagen (gray curve). The two curves were largely similar.

46 , the viscosity of crosslinked hyaluronic acid (HA) (black □ curve) was determined by the viscosity of crosslinked hyaluronic acid (HA) with monomeric collagen (▼ curve) or fibrilized collagen (▲ curve). Compared. The viscosity for crosslinked HA with fibrotic collagen was slower than that of crosslinked HA with monomeric collagen, but greater than that of crosslinked HA alone. The concentration was as in FIG. 44.

As shown in Fig . 47 , the viscosity of crosslinked hyaluronic acid (HA) was compared with the viscosity of the formulation of double crosslinked hyaluronic acid (HA)-collagen (gray curve). The viscosity for double crosslinked HA-collagen was greater than that of crosslinked HA alone.

Adding monomeric or fibrilized, cross-linked or non-crosslinked rh collagen to the crosslinked HA dermal filler did not significantly increase the expression power, enabling similar performance to the physician, but on the other hand, the viscosity of the material was significant. Increased to allow better skin lifting upon injection.

Example 22. Transdermal polymerization of recombinant human collagen methacrylate (rh collagen MA).

Recombinant human collagen with Eosin Y/TEA as a photoinitiator, injected under a mouse skin patch ( FIG. 48A ), by transmitting the skin with LED white light from a white LED torch for 6 minutes, as shown in FIGS. 48A-48B . A liquid solution of methacrylate (rh collagen MA) was subjected to transdermal polymerization. The rh collagen MA was polymerized and integrated into the skin tissue ( FIG. 48B ).

When transmitted for 6 minutes with a small LED torch in the presence of Eosin Y/TEA as a photoinitiator, rh collagen MA polymerizes under the skin.

Example 23. Preparation of double crosslinked dermal filler: 2-step synthesis

Purpose: To develop an injectable dermal filler for use in enhancing the appearance of the skin surface for aesthetic or clinical purposes. The dermal filler consists of type I recombinant human collagen (rh collagen) or a modified form thereof, methacrylated rh collagen (MA-rhCol) and crosslinked hyaluronic acid (HA).

The double-cross-linked product ( FIG. 49 ), in which cross-linked-HA is further cross-linked to rh collagen, is designed such that hyaluronic acid becomes a scaffold that provides structural support and void filling, while rh collagen enhances cell proliferation and tissue regeneration. Promote The scaffold will eventually break down, leaving behind newly formed tissue. Another objective is to analyze double crosslinked dermal fillers by examining the lifting effect (tissue strengthening) provided by crosslinking-HA, with tissue regeneration promoted by type I rh collagenase.

Way:

Double crosslinking

-HA crosslinking-

High molecular weight hyaluronic acid (range 700 KDa to 3 MDa, preferably 1.5 MDa) under alkaline conditions (e.g., pH 12 to 13 in 0.3 N Na(OH)) from 50 mg/ml to 200 mg/ml (preferably It was dissolved at a concentration in the range of 100 mg/ml). Before dissolving HA, the crosslinking agent 1, 4-butanediol diglycidyl ether (BDDE) was added to the solvent in a ratio ranging from 1% to 50% (preferably 6%, 8%, 10%) of the amount of HA disaccharide. . In some embodiments of such formulations, the HA comprises methacrylated-HA (MA-HA).

HA crosslinking was carried out at room temperature for 24 hours.

Addition and neutralization of lower MW HA

10 mg/ml to 100 mg of lower molecular weight HA (50 KDa to 1,000 KDa, preferably 300 kDa to 700 KDa) ranging from 1% to 30% (preferably 5% to 10%) of the total amount of HA It was dissolved in water at a concentration in the range of /ml (preferably 30 mg/ml). In some embodiments of such formulations, the HA comprises methacrylated-HA (MA-HA).

Before mixing the non-crosslinked HA with the crosslinked HA, HCl is added to the non-crosslinked HA in the amount required to neutralize the pH of the crosslinked HA. Phosphate buffer (PB) and NaCl are added to a final concentration of 0.1 M PB and 0.2 M NaCl.

neutralization of rh collagen

Before mixing rh-collagen with HA, make rh-collagen to 0.1 M in PB+0.2M NaCl.

Mixture of HA+rh collagen

HA (crosslinked HA + non-crosslinked HA) (6:1, 5:1, 4:1, 3:1, 2:1, 1:1,1:2, 1:3,1:4,1: 5, 1: 6) and mixed with rh collagen at a ratio of HA:rh collagen in the range between 2°C and 8°C. The final concentration of HA is about 5-50 mg/ml. The final concentration of rh collagen or MA-rh collagen is about 1 mg/ml to 50 mg/ml.

Second bridge

When HA and rh collagen are well mixed, the second crosslinking was carried out with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide metiodide (EDC): 10 to 100 times (preferably The amount of free amine in EDC and rh collagen in an amount equal to 50 times) was dissolved in (0.1 M PB+0.2 M NaCl), added to the crosslinked HA-rh collagen mixture, and mixed. The second crosslinking is performed in a dark room at 2°C to 8°C for 2 to 3 hours.

dialysis

Thereafter, the double-crosslinked material was mixed with PBS, 1 mM HCl or a low concentration phosphate buffer (low concentration phosphate buffer formulation: (a) stock solution: 10 N Na (OH) to pH 11.2 made 162 mM sodium phosphate dibasic ); (b) dialyzed against 1:1000 diluted stock solution in 0.1 mM HCL.

Rheological and mechanical evaluation

For example, the storage factor and loss factor were measured using a HAAKE-RHEO STRESS 600 ^TM instrument (THERMO SCIENTIFIC ^TM ) using a cone (1-degree) versus plate arrangement (C35/1). Frequency sweep measurements were performed over a constant strain (eg 0.8%) and frequency range (eg 0.02 Hz to 100 Hz). Optionally, various ratios or crosslinking ratios of one or both components were tested. By tuning the first crosslinking and the second crosslinking, the storage coefficient and loss factor of the final product can be adjusted.

For example, a MULTITEST 1-/i MECMESIN ^™ machine was used to measure injectability as a function of plunger displacement (mm) to observe the expression force.

Injectable

As described above, injectability measurements were performed using the MULTITEST 1-/i MECMESIN ^™ machine. A 1 ml LUER-LOK ^™ syringe (BECTON-DICKINSON ^™ ) and a 30 G needle were used for formulations 2, 2A and 3 (Table 8). The expression power as a function of plunger displacement of representative double crosslinked formulations 2, 2A and 3 (Table 8) was compared to commercially available dermal fillers likewise using a 30 G needle.

Animal research

200 microliters of Formulation 2 or 2A, or control, were injected subcutaneously into the back of Sprague dawley rats. Histology was performed after 1 week.

result:

Those skilled in the art will understand that the two-step double crosslinking herein uses a two-type crosslinking agent. The first step comprises HA and BDDE as crosslinking agents. In the second step, collagen and non-crosslinked HA are added and crosslinking is achieved using EDC. Compared to all other dermal filler manufacturing methods, it is expected that differences in crosslinking chemistry and order of action should result in dermal filler compositions having different properties including mechanical properties, tissue interactions, and rate of degradation.

A formulation of HA:rh collagen was prepared using the above method along with the representative formulations shown in Table 8.

Rheological and mechanical evaluation

As shown in Figure 50 , the storage coefficient (solid line) and loss factor (dashed line) of a representative double crosslinked formulation (Table 8) were similar to those of commercially available dermal fillers. A comparison of the storage modulus and modulus of elasticity of these formulations and these commercial fillers at f=1 Hz is shown in Figure 51 .

By tuning the first crosslinking and the second crosslinking, the storage coefficient and loss factor of the final product can be adjusted. As shown in Figure 52 , the expression power required to inject the double crosslinked formulation through a 30G needle was significantly lower than that required to inject a commercially available dermal filler.

Histology and Animal Studies

Animal studies were performed as described above. Formulations 2 and 2A (see Tables 8 and 9) were compared to commercially available dermal filler products after subcutaneous injection. Inflammation is the first step in the regenerative process, unless it is too severe.

The average histology score on day 7 after subcutaneous injection is compared to the commercially available dermal fillers in Table 9.

As shown in Table 9, the double crosslinked formulations have higher fibrosis scores and higher levels of inflammation than commercially available dermal fillers, indicating a more advanced process of tissue regeneration.

Figure 56 shows representative histological images of day 7 after subcutaneous injection of Formulations 2, 2A and controls. Arrows indicate an enhanced immune response in Formulations 2 and 2A (but still not severe), indicating the onset of tissue regeneration. " Blister" refers to a bullae formed by the injected material.

The histology score for the sample shown in FIG. 56 is presented as a bar graph in FIG. 57 , where the higher inflammation score and fibrosis score for the double crosslinked formulations 2 and 2A showed that these formulations demonstrated improved tissue regeneration compared to the control. Show.

Summary/Conclusion

The double crosslinked formulation was developed to have easy injection through 27 G to 32 G needles and a wide range of rigid G'-G". Histology results after 1-week injection show enhanced onset of the tissue regeneration process.

Example 24. Photocurable dermal filler

Purpose: To analyze the properties of photocurable dermal fillers. The photocurable formulation is half IPN before curing and ends with IPN (inter-transmitted network) after curing. It means two entangled networks, each one is crosslinked to itself and not the other.

Way:

A mixture of rh collagen and methacrylated rh collagen was added to the already crosslinked HA crosslinked as in Example 23 at a final concentration of 1 mg/ml to 10 mg/ml, wherein the methacrylated rh collagen : The ratio of non-methacrylated rh collagen is 1:0, 1:1, 1:2, 1:3, 1:4, 0:1, 2:1, 3:1, or 4:1 . The final concentration range of MA-rh collagen is about 0 mg/ml to 12 mg/ml, and the final concentration range of non-modified rh collagen is about 0 mg/ml to 12 mg/ml. The ratio of crosslinked HA: MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. The final concentration of HA is between 12 mg/ml and 25 mg/ml, and the final concentration of rh collagen (MA + non-modified) is between 1 mg/ml and 24 mg/ml.

A visible light photoinitiator was added to the mixture (eg, a composition of eosin Y, triethanolamine and N-vinylpyrrolidone).

Rheological study

1.6 ml samples of each of the representative formulations 4, 5 and 6 and the control (see Table 10 below) were poured into a cylindrical mold and a cylinder having a diameter of 2 cm and a height of 0.5 mm, and a constant amount of a white LED flashlight for 6 minutes It was cured by visible light transmission.

A formulation of highly crosslinked hyaluronic acid (HA) was mixed with a combination of three different representative ratios of rh collagen and/or rH collagen methacrylate with a constant amount of visible light photoinitiator, as shown in Table 10. . Highly BDDE crosslinked HA (but there may likewise be any other crosslinking agents, or even standard commercial fillers made only of crosslinked HA) were mixed with rhCol and rhColMA in different ratios. The result is crosslinking of HA and entangled rhColMA after curing. This form forms an interpenetrating network in which HA crosslinks to itself and collagen crosslinks to itself within the HA network.

The storage coefficient and loss coefficient were measured before and after transmission as described below.

a. Before curing

The storage factor and loss factor were measured using a HAAKE-RHEO STRESS 600 ^™ instrument (THERMO SCIENTIFIC ^™ ) using a cone (1°) versus plate arrangement (C35/1). Frequency sweep measurements were performed at a constant strain of 0.8% and a frequency ranging from 0.02 Hz to 100 Hz.

b. After curing

The storage and loss coefficients of the photocured cylinders were measured using a HAAKE-RHEO STRESS 600 ^™ instrument (THERMO SCIENTIFIC ^™ ) using a serrated plate to plate arrangement (PP20). Frequency sweep measurements were performed under a constant shear rate of 3 Pa, a constant normal load of 0.3 N at a frequency ranging from 0.02 Hz to 100 Hz.

Injectable

As described above, injectability measurements were performed using the MULTITEST 1-/ i MECMESIN ^™ machine for formulations 4, 5 and 6 and for highly crosslinked HA as a control. A 1 ml LUER-LOK ^™ syringe (BECTON-DICKINSON ^™ ) and a 30 G needle were used for all samples (Table 10). Expression power as a function of plunger displacement (12 mm/min) of representative formulations 4, 5 and 6 (Table 10) was compared with highly crosslinked HA.

Animal research

Animal studies were performed as described above. After subcutaneous injection into the rat's back, Formulation 4 (see Tables 10 and 11) was compared to the highly crosslinked HA.

result:

Rheological and mechanical evaluation

Figure 53 shows a comparison of storage and loss factors before and after photocuring formulations 4, 5 and 6 with highly crosslinked HA (see Table 10). at a frequency of f = 1 Hz, in both the before and after photo-curing, comparing the storage modulus and the elastic modulus of these agents, and (not available curing) highly cross-linked HA is shown in Figure 54. Arrows indicate “trend”: stiffness increases as the amount of rhColMA increases.

Injectable

As shown in Figure 55 , the expression power required for injection of formulations 4, 5 and 6 through a 30 G needle was lower than that required for crosslinked HA alone, which is easier to use for doctors and fine wrinkles in patients. And easier injection in the soft area. However, after in-situ photocuring (after injection), the material stiffness can be adjusted to be significantly higher than the crosslinked HA alone ( FIGS. 53 and 54 ).

Histology and Animal Studies

The average histology score of Formulation 4 upon subcutaneous injection on day 7 was compared to the highly crosslinked HA in Table 11.

As shown in Table 11, Formulation 4 has a higher fibrosis score and a higher level of inflammation than highly crosslinked HA, indicating a more advanced process of tissue regeneration.

58 and 59 show that Formulation 4 has a higher inflammation score and fibrosis score than the control dermal filler, which indicates the improved initiation of the tissue regeneration process when the dermal filler of Formulation 4 is used.

Conclusion/Summary

A photocurable filler has been developed that has relatively low stiffness before injection and allows easy injection through a 27 G to 32 G needle, but allows a significant improvement in stiffness (tunable) after photocuring. Stiffness can be tuned by adjusting the final ratio between rhCol and rhColMA. This technique allows the surgeon to shape the filler into the desired shape before fixing it with a photocurable transmissive. The injected material strongly adheres to the surrounding tissue. Preliminary in vivo results indicate the onset of the regenerative process.

Example 25. In vivo Animal Study: Independent Injection of Dermal Filler Components

Purpose: HA or its methacrylated derivative, and methacrylated rh collagen are separately injected subcutaneously in a semi-liquid phase, and after the injection, they are crosslinked in situ by white light transmission through the skin (crosslinking is rhColMA Is rhColMA). This approach allows easier scanning of the material and in -situ shaping of its shape just prior to immobilization of the material by photopolymerization. The subcutaneous rat model will be used to evaluate the characteristics of cell proliferation, tissue strengthening, and matrix degradation over time.

Method: In this model, a sample formulation component for evaluation (HA or a methacrylated derivative thereof, and a methacrylated rh collagen and photoinitiator) was injected subcutaneously on the back of a Sprague Dawley rat, and the injection site was 20 days or less. I will follow up while. The injection will be performed at approximately the same time (immediately after another injection) and at the same location. The component solution can be massaged in-situ before or simultaneously or after crosslinking. The subcutaneous rat model is chosen as it is the simplest model for estimating biocompatibility, lifting effect and persistence. Moreover, Hillel et al. published a confirmatory study for this particular model (Dermatol Surg 2012; 38:471-478).

Animals will be sedated with ketamine/Xylasine prior to each treatment. The animal's back will be shaved and the injection site will be marked on the shaved skin. To each rat, a total of 6 injections per rat will be injected with 0.2 ml of the formulation using 27.5 G to 32 G needles in spaced locations on the dorsal plane.

After injection, the formulation will be crosslinked by transdermally transmitting the injection site for 2 minutes with a white light LED torch.

Animals will be observed twice daily for morbidity and mortality throughout the study period. Every 3 days (0, 1, 4, 7, 11, 14, 18 and 21 days after injection), the height, width and length of each blister will be measured with a caliper, and the oval volume of each blister [( 4 /3)(π)(1/2 height)(1/2 length)(1/2 width)].

For example, but not limited to, animals will be euthanized on days 7, 14 and 21 post-treatment.

At each euthanasia time point, the injection site will be exposed and evaluated macroscopically, and blisters will be collected for histological evaluation. The injection site (including blisters) will be incised with the overlying skin, fixed in 4% formalin, and embedded in paraffin.

histology

Slide manufacturing

Paraffin blocks are cut to approximately 3 microns to 5 microns thick, placed on a glass slide, stained with hematoxylin and eosin (H&E) and Masson tricolor, and will be coated by an automated machine. Histological evaluation of all slides will be performed using a light microscope (Olympus BX60, serial number 7D04032).

The image will be taken at a magnification of X4. Image acquisition will be performed only upon pathological changes and only in representative animals.

result:

Results similar to those obtained in Example 24 are expected, where separate independent injections of the components of the photocurable dermal filler increased ease of injection due to, for example, the lowered viscosity of the components compared to the formulation mix. Can provide.

Although dermal fillers, including cellular growth promoting scaffolds, and uses thereof have been described with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications and variations will be apparent. Accordingly, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications and GenBank accession numbers mentioned herein are in their entirety to the same extent as if each individual publication, patent or patent application or GenBank accession number was specifically and individually incorporated by reference herein. Incorporated by reference in the specification. Moreover, citation or identification of any reference in this application should not be taken as an admission that such reference is available as prior art.

SEQUENCE LISTING <110> SHOSEYOV, Oded ORR, Nadav SEROR MAKNOUZ, Jasmine ZARKA, Revital <120> DERMAL FILLERS AND METHODS OF USE THEREOF <130> P-578412-PC <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 4662 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Collagen alpha 1(I) chain and flanking regions <400> 1 gcgatgcatg taatgtcatg agccacatga tccaatggcc acaggaacgt aagaatgtag 60 atagatttga ttttgtccgt tagatagcaa acaacattat aaaaggtgtg tatcaatacg 120 aactaattca ctcattggat tcatagaagt ccattcctcc taagtatcta aaccatggct 180 cacgctcgtg ttctcctcct cgctctcgct gttttggcaa cagctgctgt ggctgtggct 240 tctagttctt cttttgctga ttcaaaccct attagacctg ttactgatag agcagcttcc 300 actttggctc aattgcaaga ggagggccag gttgagggcc aagatgagga tatccctcca 360 attacatgcg tgcaaaatgg cttgcgttac cacgataggg atgtgtggaa acctgaacct 420 tgtcgtatct gtgtgtgtga taacggcaag gtgctctgcg atgatgttat ctgcgatgag 480 acaaaaaatt gccctggcgc tgaagttcct gagggcgagt gttgccctgt gtgccctgat 540 ggttccgagt ccccaactga tcaggaaact actggcgtgg agggcccaaa aggagatact 600 ggtccacgtg gtcctagggg tccagcaggt cctccaggta gagatggtat tccaggccag 660 cctggattgc caggaccacc aggcccacct ggcccaccag gacctcctgg tcttggtgga 720 aatttcgctc cacaactctc ttatggctat gatgagaagt caacaggtgg tatttccgtt 780 ccaggtccta tgggaccatc cggaccaaga ggtctcccag gtcctccagg tgctcctgga 840 cctcaaggct ttcaaggacc tccaggcgaa ccaggagaac caggcgcttc tggaccaatg 900 ggcccaaggg gaccacctgg cccaccagga aaaaatggcg atgatggcga agctggaaag 960 cctggtcgtc ctggagagag aggtcctcct ggcccacagg gtgcaagagg cttgccagga 1020 actgctggct tgcctggaat gaagggacat aggggcttct ccggcctcga tggcgctaag 1080 ggtgatgctg gccctgctgg accaaagggc gagccaggtt cccctggaga aaacggtgct 1140 cctggacaaa tgggtcctcg tggacttcca ggagaaaggg gtcgtccagg cgctccagga 1200 ccagcaggtg ctaggggaaa cgatggtgca acaggcgctg ctggccctcc tggcccaact 1260 ggtcctgctg gccctccagg attcccaggc gcagttggag ctaaaggaga agcaggacca 1320 cagggcccta ggggttctga aggacctcag ggtgttagag gtgaaccagg tcctccaggc 1380 ccagctggag cagctggtcc agcaggaaat ccaggtgctg atggtcaacc tggagctaag 1440 ggcgctaatg gcgcaccagg tatcgcaggc gcaccaggtt ttcctggcgc tagaggccca 1500 agtggtcctc aaggaccagg tggaccacca ggtccaaaag gcaattctgg cgaacctggc 1560 gctccaggtt ctaaaggaga tactggtgct aaaggcgaac caggacctgt tggtgttcag 1620 ggtcctcctg gtcctgctgg agaagaagga aaaagaggtg ctcgtggaga accaggacca 1680 actggacttc ctggacctcc tggtgaacgt ggcggacctg gctcaagggg tttccctgga 1740 gctgatggag tggcaggtcc aaaaggccct gctggagaga gaggttcacc aggtccagct 1800 ggtcctaagg gctcccctgg tgaagcaggt agaccaggcg aagcaggatt gccaggcgca 1860 aagggattga caggctctcc tggtagtcct ggcccagatg gaaaaacagg cccaccaggt 1920 ccagcaggac aagatggacg tccaggccca ccaggtcctc ctggagcaag gggacaagct 1980 ggcgttatgg gttttccagg acctaaaggt gctgctggag agccaggaaa ggcaggtgaa 2040 agaggagttc ctggtccacc aggagcagtg ggtcctgctg gcaaagatgg tgaagctgga 2100 gcacagggcc ctccaggccc tgctggccca gctggcgaac gtggagaaca aggcccagct 2160 ggtagtccag gatttcaagg attgcctggc cctgctggcc ctccaggaga agcaggaaaa 2220 cctggagaac aaggagttcc tggtgatttg ggagcacctg gaccttcagg agcacgtggt 2280 gaaagaggct tccctggcga gaggggtgtt caaggtccac caggtccagc aggacctaga 2340 ggtgctaatg gcgctcctgg caacgatgga gcaaaaggtg atgctggtgc tcctggcgca 2400 cctggaagtc agggtgctcc tggattgcaa ggaatgcctg gagagagggg tgctgctggc 2460 ttgccaggcc caaagggcga taggggtgat gctggaccaa aaggtgctga tggatcccca 2520 ggaaaagatg gagttcgtgg tcttactggc ccaatcggac ctccaggccc tgctggcgct 2580 ccaggtgata agggcgaaag tggcccaagt ggacctgctg gacctactgg tgctagaggt 2640 gcacctggtg ataggggtga acctggacca cctggtccag ctggttttgc tggtcctcct 2700 ggagctgatg gacaacctgg cgcaaagggt gaaccaggtg atgctggcgc aaagggagat 2760 gctggtccac ctggacctgc tggtccagca ggcccccctg ggccaatcgg taatgttgga 2820 gcaccaggtg ctaagggagc taggggttcc gctggtccac ctggagcaac aggatttcca 2880 ggcgctgctg gtagagttgg cccaccaggc ccatccggaa acgcaggccc tcctggtcct 2940 ccaggtcctg ctggcaagga gggtggcaaa ggaccaaggg gcgaaactgg ccctgctggt 3000 agacctggcg aagttggccc tcctggacca ccaggtccag caggagaaaa aggttcccca 3060 ggagctgatg gcccagctgg tgctccagga actccaggcc ctcaaggtat tgctggacag 3120 agaggcgttg tgggactccc tggtcaaagg ggagagagag gatttccagg cttgccagga 3180 cctagtggag aacctggaaa acaaggccca tcaggcgcta gtggagagcg tggacctcct 3240 ggccctatgg gacctcctgg attggctggc ccacctggcg aatcaggtcg tgaaggcgca 3300 ccaggcgcag aaggatcacc tggaagagat ggatcccctg gtgctaaagg cgatcgtgga 3360 gaaactggtc cagcaggccc accaggcgca ccaggtgcac ctggcgctcc aggacctgtg 3420 ggaccagctg gaaaatccgg agataggggc gagacaggcc cagcaggacc agctggacct 3480 gttggccctg ctggcgctcg tggaccagca ggacctcaag gaccaagggg agataaggga 3540 gaaacaggcg aacaaggcga taggggcatt aagggtcata ggggttttag tggcctccag 3600 ggtcctcctg gcccacctgg atcaccagga gaacagggac catctggtgc ttccggccca 3660 gctggtccaa gaggacctcc aggatcagct ggtgcacctg gaaaagatgg tcttaacggt 3720 ctcccaggac caatcggccc tccaggacct agaggaagaa caggagatgc tggccctgtt 3780 ggccctccag gacctcctgg tccaccaggt ccacctggtc ctccatcagc tggattcgat 3840 ttttcatttc ttccacagcc accacaagag aaagctcacg atggcggcag atattaccgt 3900 gctgatgatg ctaacgttgt tagggataga gatttggaag tggatacaac tttgaaatcc 3960 ctctcccagc aaattgaaaa cattagatct ccagaaggtt cacgtaaaaa cccagctaga 4020 acatgtcgtg atttgaaaat gtgtcactcc gattggaaaa gtggtgaata ctggattgat 4080 ccaaatcagg gctgtaatct cgatgctatc aaagttttct gtaacatgga aacaggcgaa 4140 acatgcgttt atcctactca accttccgtg gctcagaaaa attggtacat ctcaaaaaat 4200 cctaaagata agaggcacgt ttggttcggt gaaagtatga ctgatggatt tcaatttgag 4260 tacggcggtc aaggtagtga tccagctgat gtggctattc aactcacatt tttgcgtctt 4320 atgtccacag aggcatcaca aaacatcact taccactgca aaaacagtgt ggcttatatg 4380 gatcaacaaa caggaaacct taagaaggct cttcttttga agggctcaaa cgagattgag 4440 attagagcag agggcaactc aaggtttact tattcagtta ctgttgatgg ctgcacttca 4500 catactggcg cttggggtaa aacagttatc gagtataaga ctacaaaaac atcaagactc 4560 ccaatcattg atgttgctcc tctcgatgtt ggcgctcctg atcaagagtt cggttttgat 4620 gtgggcccag tttgtttcct ctaatgagct cgcggccgca tc 4662 <210> 2 <211> 4662 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Collagen alpha 1(I) chain and flanking regions <220> <221> CDS <222> (175)..(4644) <400> 2 gcgatgcatg taatgtcatg agccacatga tccaatggcc acaggaacgt aagaatgtag 60 atagatttga ttttgtccgt tagatagcaa acaacattat aaaaggtgtg tatcaatacg 120 aactaattca ctcattggat tcatagaagt ccattcctcc taagtatcta aacc atg 177 Met One gct cac gct cgt gtt ctc ctc ctc gct ctc gct gtt ttg gca aca gct 225 Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr Ala 5 10 15 gct gtg gct gtg gct tct agt tct tct ttt gct gat tca aac cct att 273 Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro Ile 20 25 30 aga cct gtt act gat aga gca gct tcc act ttg gct caa ttg caa gag 321 Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Gln Glu 35 40 45 gag ggc cag gtt gag ggc caa gat gag gat atc cct cca att aca tgc 369 Glu Gly Gln Val Glu Gly Gln Asp Glu Asp Ile Pro Pro Ile Thr Cys 50 55 60 65 gtg caa aat ggc ttg cgt tac cac gat agg gat gtg tgg aaa cct gaa 417 Val Gln Asn Gly Leu Arg Tyr His Asp Arg Asp Val Trp Lys Pro Glu 70 75 80 cct tgt cgt atc tgt gtg tgt gat aac ggc aag gtg ctc tgc gat gat 465 Pro Cys Arg Ile Cys Val Cys Asp Asn Gly Lys Val Leu Cys Asp Asp 85 90 95 gtt atc tgc gat gag aca aaa aat tgc cct ggc gct gaa gtt cct gag 513 Val Ile Cys Asp Glu Thr Lys Asn Cys Pro Gly Ala Glu Val Pro Glu 100 105 110 ggc gag tgt tgc cct gtg tgc cct gat ggt tcc gag tcc cca act gat 561 Gly Glu Cys Cys Pro Val Cys Pro Asp Gly Ser Glu Ser Pro Thr Asp 115 120 125 cag gaa act act ggc gtg gag ggc cca aaa gga gat act ggt cca cgt 609 Gln Glu Thr Thr Gly Val Glu Gly Pro Lys Gly Asp Thr Gly Pro Arg 130 135 140 145 ggt cct agg ggt cca gca ggt cct cca ggt aga gat ggt att cca ggc 657 Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly Arg Asp Gly Ile Pro Gly 150 155 160 cag cct gga ttg cca gga cca cca ggc cca cct ggc cca cca gga cct 705 Gln Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro 165 170 175 cct ggt ctt ggt gga aat ttc gct cca caa ctc tct tat ggc tat gat 753 Pro Gly Leu Gly Gly Asn Phe Ala Pro Gln Leu Ser Tyr Gly Tyr Asp 180 185 190 gag aag tca aca ggt ggt att tcc gtt cca ggt cct atg gga cca tcc 801 Glu Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro Ser 195 200 205 gga cca aga ggt ctc cca ggt cct cca ggt gct cct gga cct caa ggc 849 Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln Gly 210 215 220 225 ttt caa gga cct cca ggc gaa cca gga gaa cca ggc gct tct gga cca 897 Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly Pro 230 235 240 atg ggc cca agg gga cca cct ggc cca cca gga aaa aat ggc gat gat 945 Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp Asp 245 250 255 ggc gaa gct gga aag cct ggt cgt cct gga gag aga ggt cct cct ggc 993 Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro Gly 260 265 270 cca cag ggt gca aga ggc ttg cca gga act gct ggc ttg cct gga atg 1041 Pro Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly Met 275 280 285 aag gga cat agg ggc ttc tcc ggc ctc gat ggc gct aag ggt gat gct 1089 Lys Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp Ala 290 295 300 305 ggc cct gct gga cca aag ggc gag cca ggt tcc cct gga gaa aac ggt 1137 Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn Gly 310 315 320 gct cct gga caa atg ggt cct cgt gga ctt cca gga gaa agg ggt cgt 1185 Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly Arg 325 330 335 cca ggc gct cca gga cca gca ggt gct agg gga aac gat ggt gca aca 1233 Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala Thr 340 345 350 ggc gct gct ggc cct cct ggc cca act ggt cct gct ggc cct cca gga 1281 Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro Gly 355 360 365 ttc cca ggc gca gtt gga gct aaa gga gaa gca gga cca cag ggc cct 1329 Phe Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly Pro 370 375 380 385 agg ggt tct gaa gga cct cag ggt gtt aga ggt gaa cca ggt cct cca 1377 Arg Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro Pro 390 395 400 ggc cca gct gga gca gct ggt cca gca gga aat cca ggt gct gat ggt 1425 Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp Gly 405 410 415 caa cct gga gct aag ggc gct aat ggc gca cca ggt atc gca ggc gca 1473 Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly Ala 420 425 430 cca ggt ttt cct ggc gct aga ggc cca agt ggt cct caa gga cca ggt 1521 Pro Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro Gly 435 440 445 gga cca cca ggt cca aaa ggc aat tct ggc gaa cct ggc gct cca ggt 1569 Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro Gly 450 455 460 465 tct aaa gga gat act ggt gct aaa ggc gaa cca gga cct gtt ggt gtt 1617 Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly Val 470 475 480 cag ggt cct cct ggt cct gct gga gaa gaa gga aaa aga ggt gct cgt 1665 Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala Arg 485 490 495 gga gaa cca gga cca act gga ctt cct gga cct cct ggt gaa cgt ggc 1713 Gly Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly 500 505 510 gga cct ggc tca agg ggt ttc cct gga gct gat gga gtg gca ggt cca 1761 Gly Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly Pro 515 520 525 aaa ggc cct gct gga gag aga ggt tca cca ggt cca gct ggt cct aag 1809 Lys Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro Lys 530 535 540 545 ggc tcc cct ggt gaa gca ggt aga cca ggc gaa gca gga ttg cca ggc 1857 Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro Gly 550 555 560 gca aag gga ttg aca ggc tct cct ggt agt cct ggc cca gat gga aaa 1905 Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly Lys 565 570 575 aca ggc cca cca ggt cca gca gga caa gat gga cgt cca ggc cca cca 1953 Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro Pro 580 585 590 ggt cct cct gga gca agg gga caa gct ggc gtt atg ggt ttt cca gga 2001 Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro Gly 595 600 605 cct aaa ggt gct gct gga gag cca gga aag gca ggt gaa aga gga gtt 2049 Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly Val 610 615 620 625 cct ggt cca cca gga gca gtg ggt cct gct ggc aaa gat ggt gaa gct 2097 Pro Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu Ala 630 635 640 gga gca cag ggc cct cca ggc cct gct ggc cca gct ggc gaa cgt gga 2145 Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg Gly 645 650 655 gaa caa ggc cca gct ggt agt cca gga ttt caa gga ttg cct ggc cct 2193 Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly Pro 660 665 670 gct ggc cct cca gga gaa gca gga aaa cct gga gaa caa gga gtt cct 2241 Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val Pro 675 680 685 ggt gat ttg gga gca cct gga cct tca gga gca cgt ggt gaa aga ggc 2289 Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg Gly 690 695 700 705 ttc cct ggc gag agg ggt gtt caa ggt cca cca ggt cca gca gga cct 2337 Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly Pro 710 715 720 aga ggt gct aat ggc gct cct ggc aac gat gga gca aaa ggt gat gct 2385 Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp Ala 725 730 735 ggt gct cct ggc gca cct gga agt cag ggt gct cct gga ttg caa gga 2433 Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln Gly 740 745 750 atg cct gga gag agg ggt gct gct ggc ttg cca ggc cca aag ggc gat 2481 Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly Asp 755 760 765 agg ggt gat gct gga cca aaa ggt gct gat gga tcc cca gga aaa gat 2529 Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys Asp 770 775 780 785 gga gtt cgt ggt ctt act ggc cca atc gga cct cca ggc cct gct ggc 2577 Gly Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly 790 795 800 gct cca ggt gat aag ggc gaa agt ggc cca agt gga cct gct gga cct 2625 Ala Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly Pro 805 810 815 act ggt gct aga ggt gca cct ggt gat agg ggt gaa cct gga cca cct 2673 Thr Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro Pro 820 825 830 ggt cca gct ggt ttt gct ggt cct cct gga gct gat gga caa cct ggc 2721 Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro Gly 835 840 845 gca aag ggt gaa cca ggt gat gct ggc gca aag gga gat gct ggt cca 2769 Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly Pro 850 855 860 865 cct gga cct gct ggt cca gca ggc ccc cct ggg cca atc ggt aat gtt 2817 Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn Val 870 875 880 gga gca cca ggt gct aag gga gct agg ggt tcc gct ggt cca cct gga 2865 Gly Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro Gly 885 890 895 gca aca gga ttt cca ggc gct gct ggt aga gtt ggc cca cca ggc cca 2913 Ala Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly Pro 900 905 910 tcc gga aac gca ggc cct cct ggt cct cca ggt cct gct ggc aag gag 2961 Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu 915 920 925 ggt ggc aaa gga cca agg ggc gaa act ggc cct gct ggt aga cct ggc 3009 Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro Gly 930 935 940 945 gaa gtt ggc cct cct gga cca cca ggt cca gca gga gaa aaa ggt tcc 3057 Glu Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly Ser 950 955 960 cca gga gct gat ggc cca gct ggt gct cca gga act cca ggc cct caa 3105 Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro Gln 965 970 975 ggt att gct gga cag aga ggc gtt gtg gga ctc cct ggt caa agg gga 3153 Gly Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg Gly 980 985 990 gag aga gga ttt cca ggc ttg cca gga cct agt gga gaa cct gga aaa 3201 Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly Lys 995 1000 1005 caa ggc cca tca ggc gct agt gga gag cgt gga cct cct ggc cct 3246 Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly Pro 1010 1015 1020 atg gga cct cct gga ttg gct ggc cca cct ggc gaa tca ggt cgt 3291 Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly Arg 1025 1030 1035 gaa ggc gca cca ggc gca gaa gga tca cct gga aga gat gga tcc 3336 Glu Gly Ala Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly Ser 1040 1045 1050 cct ggt gct aaa ggc gat cgt gga gaa act ggt cca gca ggc cca 3381 Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro 1055 1060 1065 cca ggc gca cca ggt gca cct ggc gct cca gga cct gtg gga cca 3426 Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Val Gly Pro 1070 1075 1080 gct gga aaa tcc gga gat agg ggc gag aca ggc cca gca gga cca 3471 Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly Pro 1085 1090 1095 gct gga cct gtt ggc cct gct ggc gct cgt gga cca gca gga cct 3516 Ala Gly Pro Val Gly Pro Ala Gly Ala Arg Gly Pro Ala Gly Pro 1100 1105 1110 caa gga cca agg gga gat aag gga gaa aca ggc gaa caa ggc gat 3561 Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp 1115 1120 1125 agg ggc att aag ggt cat agg ggt ttt agt ggc ctc cag ggt cct 3606 Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro 1130 1135 1140 cct ggc cca cct gga tca cca gga gaa cag gga cca tct ggt gct 3651 Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala 1145 1150 1155 tcc ggc cca gct ggt cca aga gga cct cca gga tca gct ggt gca 3696 Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala 1160 1165 1170 cct gga aaa gat ggt ctt aac ggt ctc cca gga cca atc ggc cct 3741 Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro 1175 1180 1185 cca gga cct aga gga aga aca gga gat gct ggc cct gtt ggc cct 3786 Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro 1190 1195 1200 cca gga cct cct ggt cca cca ggt cca cct ggt cct cca tca gct 3831 Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala 1205 1210 1215 gga ttc gat ttt tca ttt ctt cca cag cca cca caa gag aaa gct 3876 Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys Ala 1220 1225 1230 cac gat ggc ggc aga tat tac cgt gct gat gat gct aac gtt gtt 3921 His Asp Gly Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Val Val 1235 1240 1245 agg gat aga gat ttg gaa gtg gat aca act ttg aaa tcc ctc tcc 3966 Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu Ser 1250 1255 1260 cag caa att gaa aac att aga tct cca gaa ggt tca cgt aaa aac 4011 Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys Asn 1265 1270 1275 cca gct aga aca tgt cgt gat ttg aaa atg tgt cac tcc gat tgg 4056 Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp Trp 1280 1285 1290 aaa agt ggt gaa tac tgg att gat cca aat cag ggc tgt aat ctc 4101 Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn Leu 1295 1300 1305 gat gct atc aaa gtt ttc tgt aac atg gaa aca ggc gaa aca tgc 4146 Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr Cys 1310 1315 1320 gtt tat cct act caa cct tcc gtg gct cag aaa aat tgg tac atc 4191 Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr Ile 1325 1330 1335 tca aaa aat cct aaa gat aag agg cac gtt tgg ttc ggt gaa agt 4236 Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu Ser 1340 1345 1350 atg act gat gga ttt caa ttt gag tac ggc ggt caa ggt agt gat 4281 Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp 1355 1360 1365 cca gct gat gtg gct att caa ctc aca ttt ttg cgt ctt atg tcc 4326 Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser 1370 1375 1380 aca gag gca tca caa aac atc act tac cac tgc aaa aac agt gtg 4371 Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val 1385 1390 1395 gct tat atg gat caa caa aca gga aac ctt aag aag gct ctt ctt 4416 Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu 1400 1405 1410 ttg aag ggc tca aac gag att gag att aga gca gag ggc aac tca 4461 Leu Lys Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser 1415 1420 1425 agg ttt act tat tca gtt act gtt gat ggc tgc act tca cat act 4506 Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His Thr 1430 1435 1440 ggc gct tgg ggt aaa aca gtt atc gag tat aag act aca aaa aca 4551 Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys Thr 1445 1450 1455 tca aga ctc cca atc att gat gtt gct cct ctc gat gtt ggc gct 4596 Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly Ala 1460 1465 1470 cct gat caa gag ttc ggt ttt gat gtg ggc cca gtt tgt ttc ctc 4641 Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe Leu 1475 1480 1485 taa tgagctcgcg gccgcatc 4662 <210> 3 <211> 1489 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Gln 35 40 45 Glu Glu Gly Gln Val Glu Gly Gln Asp Glu Asp Ile Pro Pro Ile Thr 50 55 60 Cys Val Gln Asn Gly Leu Arg Tyr His Asp Arg Asp Val Trp Lys Pro 65 70 75 80 Glu Pro Cys Arg Ile Cys Val Cys Asp Asn Gly Lys Val Leu Cys Asp 85 90 95 Asp Val Ile Cys Asp Glu Thr Lys Asn Cys Pro Gly Ala Glu Val Pro 100 105 110 Glu Gly Glu Cys Cys Pro Val Cys Pro Asp Gly Ser Glu Ser Pro Thr 115 120 125 Asp Gln Glu Thr Thr Gly Val Glu Gly Pro Lys Gly Asp Thr Gly Pro 130 135 140 Arg Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly Arg Asp Gly Ile Pro 145 150 155 160 Gly Gln Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 165 170 175 Pro Pro Gly Leu Gly Gly Asn Phe Ala Pro Gln Leu Ser Tyr Gly Tyr 180 185 190 Asp Glu Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro 195 200 205 Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln 210 215 220 Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly 225 230 235 240 Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp 245 250 255 Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro 260 265 270 Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly 275 280 285 Met Lys Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp 290 295 300 Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn 305 310 315 320 Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly 325 330 335 Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala 340 345 350 Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro 355 360 365 Gly Phe Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly 370 375 380 Pro Arg Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro 385 390 395 400 Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp 405 410 415 Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly 420 425 430 Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro 435 440 445 Gly Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro 450 455 460 Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly 465 470 475 480 Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala 485 490 495 Arg Gly Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg 500 505 510 Gly Gly Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly 515 520 525 Pro Lys Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro 530 535 540 Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro 545 550 555 560 Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly 565 570 575 Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro 580 585 590 Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro 595 600 605 Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly 610 615 620 Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu 625 630 635 640 Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg 645 650 655 Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly 660 665 670 Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val 675 680 685 Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg 690 695 700 Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly 705 710 715 720 Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp 725 730 735 Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln 740 745 750 Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly 755 760 765 Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys 770 775 780 Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala 785 790 795 800 Gly Ala Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly 805 810 815 Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro 820 825 830 Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro 835 840 845 Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly 850 855 860 Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn 865 870 875 880 Val Gly Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro 885 890 895 Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly 900 905 910 Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys 915 920 925 Glu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro 930 935 940 Gly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly 945 950 955 960 Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro 965 970 975 Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg 980 985 990 Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly 995 1000 1005 Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly 1010 1015 1020 Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly 1025 1030 1035 Arg Glu Gly Ala Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly 1040 1045 1050 Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly 1055 1060 1065 Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Val Gly 1070 1075 1080 Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly 1085 1090 1095 Pro Ala Gly Pro Val Gly Pro Ala Gly Ala Arg Gly Pro Ala Gly 1100 1105 1110 Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly 1115 1120 1125 Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly 1130 1135 1140 Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly 1145 1150 1155 Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly 1160 1165 1170 Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly 1175 1180 1185 Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly 1190 1195 1200 Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser 1205 1210 1215 Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys 1220 1225 1230 Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Val 1235 1240 1245 Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu 1250 1255 1260 Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys 1265 1270 1275 Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp 1280 1285 1290 Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn 1295 1300 1305 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr 1310 1315 1320 Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr 1325 1330 1335 Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu 1340 1345 1350 Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser 1355 1360 1365 Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met 1370 1375 1380 Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser 1385 1390 1395 Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu 1400 1405 1410 Leu Leu Lys Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn 1415 1420 1425 Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His 1430 1435 1440 Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys 1445 1450 1455 Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly 1460 1465 1470 Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe 1475 1480 1485 Leu <210> 4 <211> 4362 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Collagen alpha 2(I) chain and flanking regions <400> 4 gcgatgcatg taatgtcatg agccacatga tccaatggcc acaggaacgt aagaatgtag 60 atagatttga ttttgtccgt tagatagcaa acaacattat aaaaggtgtg tatcaatacg 120 aactaattca ctcattggat tcatagaagt ccattcctcc taagtatcta aaccatggct 180 cacgctcgtg ttctcctcct cgctctcgct gttttggcaa cagctgctgt ggctgtggct 240 tcaagttcta gttttgctga ttccaaccca attcgtccag ttactgatag agcagcttcc 300 actttggctc aattgcttca agaagaaact gtgaggaagg gccctgctgg cgataggggc 360 cctaggggcg aaaggggtcc accaggacct ccaggcaggg atggcgaaga tggtccaact 420 ggccctcctg gacctcctgg ccctccaggg ccacccggct tgggcggaaa cttcgcagct 480 caatacgatg gcaagggtgt tggtcttggt cctggtccta tgggcttgat gggacctaga 540 ggcccacctg gtgctgctgg tgctcctgga ccacagggtt ttcagggacc agctggcgag 600 ccaggagagc caggccaaac aggaccagct ggtgcaaggg gacctgctgg acctcctgga 660 aaagctggtg aagatggtca cccaggcaaa ccaggacgtc ctggcgaaag aggtgttgtt 720 ggaccacaag gcgctagggg atttccaggt acacctggat tgccaggttt taagggcatt 780 cgtggtcata acggcctcga tggattgaag ggacagcctg gcgcacctgg cgttaagggt 840 gaacctggag caccaggtga aaacggtact cctggccaga ctggtgcaag aggactccca 900 ggtgaaaggg gtagagttgg tgctcctgga cctgctggag ctaggggtag tgatggtagt 960 gttggtcctg tgggccctgc tggtccaatc ggttccgctg gcccacctgg attcccaggc 1020 gctccaggac ctaaaggaga aatcggtgct gtgggtaacg caggtcctac tggtccagca 1080 ggtcctcgtg gagaagtggg attgccagga ctttctggtc cagtgggccc tccaggcaac 1140 cctggagcta acggcttgac aggagctaaa ggcgcagcag gactccctgg agtggctggc 1200 gcaccaggat tgcctggtcc aaggggtatc ccaggccctg ttggcgcagc tggagctact 1260 ggtgcacgtg gacttgttgg cgaaccaggc cctgctggat caaaaggcga gtctggaaat 1320 aagggagaac ctggttctgc tggacctcaa ggtcctcctg gaccttctgg agaagaagga 1380 aaaaggggac caaatggcga ggctggatca gcaggtccac caggaccacc tggacttcgt 1440 ggatcccctg gtagtagagg acttccaggc gctgatggta gagcaggcgt tatgggacca 1500 ccaggaagta gaggagcatc cggtccagca ggagttaggg gtcctaacgg agatgctggt 1560 agaccaggtg aaccaggtct tatgggccca aggggcctcc caggtagtcc aggaaatatc 1620 ggccctgctg gaaaagaagg ccctgttgga cttccaggta ttgatggacg tcctggccct 1680 attggcccag caggtgcaag aggagaacct ggcaatattg gatttccagg accaaagggt 1740 ccaacaggcg atcctggaaa aaatggagat aagggtcatg ctggattggc aggcgcaagg 1800 ggcgctcctg gtccagatgg aaacaacggc gcacagggtc cacctggccc tcagggtgtt 1860 caaggcggaa aaggcgaaca aggcccagct ggaccaccag gctttcaagg cttgccagga 1920 ccaagtggtc cagcaggtga agttggcaag ccaggcgagc gtggacttca tggcgagttt 1980 ggactccctg gaccagcagg accaaggggt gaaagaggcc ctcctggaga gagtggcgct 2040 gctggaccaa caggcccaat cggtagtaga ggtcctagtg gacctccagg cccagatgga 2100 aataagggtg aaccaggagt tgtgggcgct gttggaacag ctggtccttc aggaccatca 2160 ggactcccag gcgagagagg cgctgctggc attcctggag gaaaaggtga aaaaggcgaa 2220 cctggcctcc gtggcgaaat cggaaatcct ggacgtgatg gtgctcgtgg tgcacacggc 2280 gctgtgggcg ctccaggccc tgctggtgct actggtgata gaggagaggc tggcgcagct 2340 ggcccagcag gtcctgctgg cccaaggggt agtcctggtg aaagaggcga agttggacct 2400 gctggcccta acggctttgc tggccctgct ggagcagcag gtcaacctgg cgctaaaggt 2460 gaaaggggcg gaaagggccc aaaaggtgaa aatggcgttg tgggaccaac tggtccagtg 2520 ggcgcagctg gacctgctgg tccaaatgga ccaccaggac cagcaggtag tagaggagat 2580 ggtggacctc caggaatgac aggttttcca ggtgctgctg gtagaacagg acctcctggt 2640 cctagtggta tttctggtcc accaggacca ccaggtcctg ctggaaaaga aggattgagg 2700 ggtccacgtg gtgatcaagg accagtgggc agaactggtg aagttggcgc agtgggacca 2760 cctggttttg ctggagaaaa gggcccttct ggagaggcag gaacagctgg tcctcctggt 2820 acacctggac ctcaaggact tttgggtgca cctggtattc tcggattgcc aggaagtagg 2880 ggcgaacgtg gacttcctgg cgtggcagga gcagttggag aacctggccc tctcggaatc 2940 gcaggcccac caggcgcaag aggaccacca ggagctgttg gatcaccagg cgtgaatggt 3000 gcacctggcg aggctggtcg tgatggaaac ccaggaaatg atggcccacc aggaagagat 3060 ggtcaacctg gacacaaagg cgagaggggc tacccaggaa atattggccc agttggtgct 3120 gctggcgcac caggcccaca cggtccagtt ggaccagcag gaaaacacgg taatcgtggc 3180 gaaacaggcc cttcaggccc agtgggacct gctggtgctg ttggcccaag aggaccatct 3240 ggacctcaag gcattagagg cgataaggga gagcctggcg aaaaaggacc tagaggcttg 3300 cctggtttta aaggacacaa cggtctccaa ggacttccag gtatcgctgg tcatcatgga 3360 gatcagggtg ctcctggatc agtgggtcca gcaggtccta gaggcccagc aggcccttcc 3420 ggtccagcag gaaaggatgg acgtactggc caccctggaa ctgtgggccc tgctggaatt 3480 agaggtcctc aaggtcatca gggccctgct ggccctccag gtccaccagg tcctccaggc 3540 ccaccaggag tttcaggtgg tggttacgat tttggttacg atggtgattt ttaccgtgct 3600 gatcaaccta gaagtgctcc ttctctccgt cctaaagatt atgaagttga tgctactttg 3660 aaatcactta acaaccagat tgagactctt ctcacacctg agggatcaag aaagaatcca 3720 gcacgtacat gccgtgatct cagacttagt cacccagagt ggtcaagtgg ctattattgg 3780 attgatccta atcagggttg tacaatggag gctatcaaag tttactgtga ttttccaact 3840 ggagagacat gtattagggc acaacctgag aacattccag ctaaaaattg gtatcgttcc 3900 tctaaagata agaaacatgt ttggctcgga gagactatta acgctggttc tcagttcgag 3960 tataatgttg agggcgttac ttctaaagag atggcaactc agctcgcttt tatgagattg 4020 ctcgctaact acgcatccca aaacatcact tatcactgca aaaattccat tgcatatatg 4080 gatgaggaga caggaaattt gaagaaagca gttattctcc aaggtagtaa cgatgttgag 4140 cttgtggctg agggaaatag tagattcact tacacagttt tggtggatgg atgctcaaag 4200 aaaactaatg agtggggcaa gacaatcatt gagtacaaga caaataagcc ttctaggctc 4260 ccatttctcg atattgcacc tcttgatatc ggaggagctg atcacgagtt ttttgttgat 4320 atcggacctg tttgttttaa gtaatgagct cgcggccgca tc 4362 <210> 5 <211> 4362 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Collagen alpha 2(I) chain and flanking regions <220> <221> CDS <222> (175)..(4344) <400> 5 gcgatgcatg taatgtcatg agccacatga tccaatggcc acaggaacgt aagaatgtag 60 atagatttga ttttgtccgt tagatagcaa acaacattat aaaaggtgtg tatcaatacg 120 aactaattca ctcattggat tcatagaagt ccattcctcc taagtatcta aacc atg 177 Met One gct cac gct cgt gtt ctc ctc ctc gct ctc gct gtt ttg gca aca gct 225 Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr Ala 5 10 15 gct gtg gct gtg gct tca agt tct agt ttt gct gat tcc aac cca att 273 Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro Ile 20 25 30 cgt cca gtt act gat aga gca gct tcc act ttg gct caa ttg ctt caa 321 Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Leu Gln 35 40 45 gaa gaa act gtg agg aag ggc cct gct ggc gat agg ggc cct agg ggc 369 Glu Glu Thr Val Arg Lys Gly Pro Ala Gly Asp Arg Gly Pro Arg Gly 50 55 60 65 gaa agg ggt cca cca gga cct cca ggc agg gat ggc gaa gat ggt cca 417 Glu Arg Gly Pro Pro Gly Pro Pro Gly Arg Asp Gly Glu Asp Gly Pro 70 75 80 act ggc cct cct gga cct cct ggc cct cca ggg cca ccc ggc ttg ggc 465 Thr Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly 85 90 95 gga aac ttc gca gct caa tac gat ggc aag ggt gtt ggt ctt ggt cct 513 Gly Asn Phe Ala Ala Gln Tyr Asp Gly Lys Gly Val Gly Leu Gly Pro 100 105 110 ggt cct atg ggc ttg atg gga cct aga ggc cca cct ggt gct gct ggt 561 Gly Pro Met Gly Leu Met Gly Pro Arg Gly Pro Pro Gly Ala Ala Gly 115 120 125 gct cct gga cca cag ggt ttt cag gga cca gct ggc gag cca gga gag 609 Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Ala Gly Glu Pro Gly Glu 130 135 140 145 cca ggc caa aca gga cca gct ggt gca agg gga cct gct gga cct cct 657 Pro Gly Gln Thr Gly Pro Ala Gly Ala Arg Gly Pro Ala Gly Pro Pro 150 155 160 gga aaa gct ggt gaa gat ggt cac cca ggc aaa cca gga cgt cct ggc 705 Gly Lys Ala Gly Glu Asp Gly His Pro Gly Lys Pro Gly Arg Pro Gly 165 170 175 gaa aga ggt gtt gtt gga cca caa ggc gct agg gga ttt cca ggt aca 753 Glu Arg Gly Val Val Gly Pro Gln Gly Ala Arg Gly Phe Pro Gly Thr 180 185 190 cct gga ttg cca ggt ttt aag ggc att cgt ggt cat aac ggc ctc gat 801 Pro Gly Leu Pro Gly Phe Lys Gly Ile Arg Gly His Asn Gly Leu Asp 195 200 205 gga ttg aag gga cag cct ggc gca cct ggc gtt aag ggt gaa cct gga 849 Gly Leu Lys Gly Gln Pro Gly Ala Pro Gly Val Lys Gly Glu Pro Gly 210 215 220 225 gca cca ggt gaa aac ggt act cct ggc cag act ggt gca aga gga ctc 897 Ala Pro Gly Glu Asn Gly Thr Pro Gly Gln Thr Gly Ala Arg Gly Leu 230 235 240 cca ggt gaa agg ggt aga gtt ggt gct cct gga cct gct gga gct agg 945 Pro Gly Glu Arg Gly Arg Val Gly Ala Pro Gly Pro Ala Gly Ala Arg 245 250 255 ggt agt gat ggt agt gtt ggt cct gtg ggc cct gct ggt cca atc ggt 993 Gly Ser Asp Gly Ser Val Gly Pro Val Gly Pro Ala Gly Pro Ile Gly 260 265 270 tcc gct ggc cca cct gga ttc cca ggc gct cca gga cct aaa gga gaa 1041 Ser Ala Gly Pro Pro Gly Phe Pro Gly Ala Pro Gly Pro Lys Gly Glu 275 280 285 atc ggt gct gtg ggt aac gca ggt cct act ggt cca gca ggt cct cgt 1089 Ile Gly Ala Val Gly Asn Ala Gly Pro Thr Gly Pro Ala Gly Pro Arg 290 295 300 305 gga gaa gtg gga ttg cca gga ctt tct ggt cca gtg ggc cct cca ggc 1137 Gly Glu Val Gly Leu Pro Gly Leu Ser Gly Pro Val Gly Pro Pro Gly 310 315 320 aac cct gga gct aac ggc ttg aca gga gct aaa ggc gca gca gga ctc 1185 Asn Pro Gly Ala Asn Gly Leu Thr Gly Ala Lys Gly Ala Ala Gly Leu 325 330 335 cct gga gtg gct ggc gca cca gga ttg cct ggt cca agg ggt atc cca 1233 Pro Gly Val Ala Gly Ala Pro Gly Leu Pro Gly Pro Arg Gly Ile Pro 340 345 350 ggc cct gtt ggc gca gct gga gct act ggt gca cgt gga ctt gtt ggc 1281 Gly Pro Val Gly Ala Ala Gly Ala Thr Gly Ala Arg Gly Leu Val Gly 355 360 365 gaa cca ggc cct gct gga tca aaa ggc gag tct gga aat aag gga gaa 1329 Glu Pro Gly Pro Ala Gly Ser Lys Gly Glu Ser Gly Asn Lys Gly Glu 370 375 380 385 cct ggt tct gct gga cct caa ggt cct cct gga cct tct gga gaa gaa 1377 Pro Gly Ser Ala Gly Pro Gln Gly Pro Pro Gly Pro Ser Gly Glu Glu 390 395 400 gga aaa agg gga cca aat ggc gag gct gga tca gca ggt cca cca gga 1425 Gly Lys Arg Gly Pro Asn Gly Glu Ala Gly Ser Ala Gly Pro Pro Gly 405 410 415 cca cct gga ctt cgt gga tcc cct ggt agt aga gga ctt cca ggc gct 1473 Pro Pro Gly Leu Arg Gly Ser Pro Gly Ser Arg Gly Leu Pro Gly Ala 420 425 430 gat ggt aga gca ggc gtt atg gga cca cca gga agt aga gga gca tcc 1521 Asp Gly Arg Ala Gly Val Met Gly Pro Pro Gly Ser Arg Gly Ala Ser 435 440 445 ggt cca gca gga gtt agg ggt cct aac gga gat gct ggt aga cca ggt 1569 Gly Pro Ala Gly Val Arg Gly Pro Asn Gly Asp Ala Gly Arg Pro Gly 450 455 460 465 gaa cca ggt ctt atg ggc cca agg ggc ctc cca ggt agt cca gga aat 1617 Glu Pro Gly Leu Met Gly Pro Arg Gly Leu Pro Gly Ser Pro Gly Asn 470 475 480 atc ggc cct gct gga aaa gaa ggc cct gtt gga ctt cca ggt att gat 1665 Ile Gly Pro Ala Gly Lys Glu Gly Pro Val Gly Leu Pro Gly Ile Asp 485 490 495 gga cgt cct ggc cct att ggc cca gca ggt gca aga gga gaa cct ggc 1713 Gly Arg Pro Gly Pro Ile Gly Pro Ala Gly Ala Arg Gly Glu Pro Gly 500 505 510 aat att gga ttt cca gga cca aag ggt cca aca ggc gat cct gga aaa 1761 Asn Ile Gly Phe Pro Gly Pro Lys Gly Pro Thr Gly Asp Pro Gly Lys 515 520 525 aat gga gat aag ggt cat gct gga ttg gca ggc gca agg ggc gct cct 1809 Asn Gly Asp Lys Gly His Ala Gly Leu Ala Gly Ala Arg Gly Ala Pro 530 535 540 545 ggt cca gat gga aac aac ggc gca cag ggt cca cct ggc cct cag ggt 1857 Gly Pro Asp Gly Asn Asn Gly Ala Gln Gly Pro Pro Gly Pro Gln Gly 550 555 560 gtt caa ggc gga aaa ggc gaa caa ggc cca gct gga cca cca ggc ttt 1905 Val Gln Gly Gly Lys Gly Glu Gln Gly Pro Ala Gly Pro Pro Gly Phe 565 570 575 caa ggc ttg cca gga cca agt ggt cca gca ggt gaa gtt ggc aag cca 1953 Gln Gly Leu Pro Gly Pro Ser Gly Pro Ala Gly Glu Val Gly Lys Pro 580 585 590 ggc gag cgt gga ctt cat ggc gag ttt gga ctc cct gga cca gca gga 2001 Gly Glu Arg Gly Leu His Gly Glu Phe Gly Leu Pro Gly Pro Ala Gly 595 600 605 cca agg ggt gaa aga ggc cct cct gga gag agt ggc gct gct gga cca 2049 Pro Arg Gly Glu Arg Gly Pro Pro Gly Glu Ser Gly Ala Ala Gly Pro 610 615 620 625 aca ggc cca atc ggt agt aga ggt cct agt gga cct cca ggc cca gat 2097 Thr Gly Pro Ile Gly Ser Arg Gly Pro Ser Gly Pro Pro Gly Pro Asp 630 635 640 gga aat aag ggt gaa cca gga gtt gtg ggc gct gtt gga aca gct ggt 2145 Gly Asn Lys Gly Glu Pro Gly Val Val Gly Ala Val Gly Thr Ala Gly 645 650 655 cct tca gga cca tca gga ctc cca ggc gag aga ggc gct gct ggc att 2193 Pro Ser Gly Pro Ser Gly Leu Pro Gly Glu Arg Gly Ala Ala Gly Ile 660 665 670 cct gga gga aaa ggt gaa aaa ggc gaa cct ggc ctc cgt ggc gaa atc 2241 Pro Gly Gly Lys Gly Glu Lys Gly Glu Pro Gly Leu Arg Gly Glu Ile 675 680 685 gga aat cct gga cgt gat ggt gct cgt ggt gca cac ggc gct gtg ggc 2289 Gly Asn Pro Gly Arg Asp Gly Ala Arg Gly Ala His Gly Ala Val Gly 690 695 700 705 gct cca ggc cct gct ggt gct act ggt gat aga gga gag gct ggc gca 2337 Ala Pro Gly Pro Ala Gly Ala Thr Gly Asp Arg Gly Glu Ala Gly Ala 710 715 720 gct ggc cca gca ggt cct gct ggc cca agg ggt agt cct ggt gaa aga 2385 Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg 725 730 735 ggc gaa gtt gga cct gct ggc cct aac ggc ttt gct ggc cct gct gga 2433 Gly Glu Val Gly Pro Ala Gly Pro Asn Gly Phe Ala Gly Pro Ala Gly 740 745 750 gca gca ggt caa cct ggc gct aaa ggt gaa agg ggc gga aag ggc cca 2481 Ala Ala Gly Gln Pro Gly Ala Lys Gly Glu Arg Gly Gly Lys Gly Pro 755 760 765 aaa ggt gaa aat ggc gtt gtg gga cca act ggt cca gtg ggc gca gct 2529 Lys Gly Glu Asn Gly Val Val Gly Pro Thr Gly Pro Val Gly Ala Ala 770 775 780 785 gga cct gct ggt cca aat gga cca cca gga cca gca ggt agt aga gga 2577 Gly Pro Ala Gly Pro Asn Gly Pro Pro Gly Pro Ala Gly Ser Arg Gly 790 795 800 gat ggt gga cct cca gga atg aca ggt ttt cca ggt gct gct ggt aga 2625 Asp Gly Gly Pro Pro Gly Met Thr Gly Phe Pro Gly Ala Ala Gly Arg 805 810 815 aca gga cct cct ggt cct agt ggt att tct ggt cca cca gga cca cca 2673 Thr Gly Pro Pro Gly Pro Ser Gly Ile Ser Gly Pro Pro Gly Pro Pro 820 825 830 ggt cct gct gga aaa gaa gga ttg agg ggt cca cgt ggt gat caa gga 2721 Gly Pro Ala Gly Lys Glu Gly Leu Arg Gly Pro Arg Gly Asp Gln Gly 835 840 845 cca gtg ggc aga act ggt gaa gtt ggc gca gtg gga cca cct ggt ttt 2769 Pro Val Gly Arg Thr Gly Glu Val Gly Ala Val Gly Pro Pro Gly Phe 850 855 860 865 gct gga gaa aag ggc cct tct gga gag gca gga aca gct ggt cct cct 2817 Ala Gly Glu Lys Gly Pro Ser Gly Glu Ala Gly Thr Ala Gly Pro Pro 870 875 880 ggt aca cct gga cct caa gga ctt ttg ggt gca cct ggt att ctc gga 2865 Gly Thr Pro Gly Pro Gln Gly Leu Leu Gly Ala Pro Gly Ile Leu Gly 885 890 895 ttg cca gga agt agg ggc gaa cgt gga ctt cct ggc gtg gca gga gca 2913 Leu Pro Gly Ser Arg Gly Glu Arg Gly Leu Pro Gly Val Ala Gly Ala 900 905 910 gtt gga gaa cct ggc cct ctc gga atc gca ggc cca cca ggc gca aga 2961 Val Gly Glu Pro Gly Pro Leu Gly Ile Ala Gly Pro Pro Gly Ala Arg 915 920 925 gga cca cca gga gct gtt gga tca cca ggc gtg aat ggt gca cct ggc 3009 Gly Pro Pro Gly Ala Val Gly Ser Pro Gly Val Asn Gly Ala Pro Gly 930 935 940 945 gag gct ggt cgt gat gga aac cca gga aat gat ggc cca cca gga aga 3057 Glu Ala Gly Arg Asp Gly Asn Pro Gly Asn Asp Gly Pro Pro Gly Arg 950 955 960 gat ggt caa cct gga cac aaa ggc gag agg ggc tac cca gga aat att 3105 Asp Gly Gln Pro Gly His Lys Gly Glu Arg Gly Tyr Pro Gly Asn Ile 965 970 975 ggc cca gtt ggt gct gct ggc gca cca ggc cca cac ggt cca gtt gga 3153 Gly Pro Val Gly Ala Ala Gly Ala Pro Gly Pro His Gly Pro Val Gly 980 985 990 cca gca gga aaa cac ggt aat cgt ggc gaa aca ggc cct tca ggc cca 3201 Pro Ala Gly Lys His Gly Asn Arg Gly Glu Thr Gly Pro Ser Gly Pro 995 1000 1005 gtg gga cct gct ggt gct gtt ggc cca aga gga cca tct gga cct 3246 Val Gly Pro Ala Gly Ala Val Gly Pro Arg Gly Pro Ser Gly Pro 1010 1015 1020 caa ggc att aga ggc gat aag gga gag cct ggc gaa aaa gga cct 3291 Gln Gly Ile Arg Gly Asp Lys Gly Glu Pro Gly Glu Lys Gly Pro 1025 1030 1035 aga ggc ttg cct ggt ttt aaa gga cac aac ggt ctc caa gga ctt 3336 Arg Gly Leu Pro Gly Phe Lys Gly His Asn Gly Leu Gln Gly Leu 1040 1045 1050 cca ggt atc gct ggt cat cat gga gat cag ggt gct cct gga tca 3381 Pro Gly Ile Ala Gly His His Gly Asp Gln Gly Ala Pro Gly Ser 1055 1060 1065 gtg ggt cca gca ggt cct aga ggc cca gca ggc cct tcc ggt cca 3426 Val Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly Pro Ser Gly Pro 1070 1075 1080 gca gga aag gat gga cgt act ggc cac cct gga act gtg ggc cct 3471 Ala Gly Lys Asp Gly Arg Thr Gly His Pro Gly Thr Val Gly Pro 1085 1090 1095 gct gga att aga ggt cct caa ggt cat cag ggc cct gct ggc cct 3516 Ala Gly Ile Arg Gly Pro Gln Gly His Gln Gly Pro Ala Gly Pro 1100 1105 1110 cca ggt cca cca ggt cct cca ggc cca cca gga gtt tca ggt ggt 3561 Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Val Ser Gly Gly 1115 1120 1125 ggt tac gat ttt ggt tac gat ggt gat ttt tac cgt gct gat caa 3606 Gly Tyr Asp Phe Gly Tyr Asp Gly Asp Phe Tyr Arg Ala Asp Gln 1130 1135 1140 cct aga agt gct cct tct ctc cgt cct aaa gat tat gaa gtt gat 3651 Pro Arg Ser Ala Pro Ser Leu Arg Pro Lys Asp Tyr Glu Val Asp 1145 1150 1155 gct act ttg aaa tca ctt aac aac cag att gag act ctt ctc aca 3696 Ala Thr Leu Lys Ser Leu Asn Asn Gln Ile Glu Thr Leu Leu Thr 1160 1165 1170 cct gag gga tca aga aag aat cca gca cgt aca tgc cgt gat ctc 3741 Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu 1175 1180 1185 aga ctt agt cac cca gag tgg tca agt ggc tat tat tgg att gat 3786 Arg Leu Ser His Pro Glu Trp Ser Ser Gly Tyr Tyr Trp Ile Asp 1190 1195 1200 cct aat cag ggt tgt aca atg gag gct atc aaa gtt tac tgt gat 3831 Pro Asn Gln Gly Cys Thr Met Glu Ala Ile Lys Val Tyr Cys Asp 1205 1210 1215 ttt cca act gga gag aca tgt att agg gca caa cct gag aac att 3876 Phe Pro Thr Gly Glu Thr Cys Ile Arg Ala Gln Pro Glu Asn Ile 1220 1225 1230 cca gct aaa aat tgg tat cgt tcc tct aaa gat aag aaa cat gtt 3921 Pro Ala Lys Asn Trp Tyr Arg Ser Ser Lys Asp Lys Lys His Val 1235 1240 1245 tgg ctc gga gag act att aac gct ggt tct cag ttc gag tat aat 3966 Trp Leu Gly Glu Thr Ile Asn Ala Gly Ser Gln Phe Glu Tyr Asn 1250 1255 1260 gtt gag ggc gtt act tct aaa gag atg gca act cag ctc gct ttt 4011 Val Glu Gly Val Thr Ser Lys Glu Met Ala Thr Gln Leu Ala Phe 1265 1270 1275 atg aga ttg ctc gct aac tac gca tcc caa aac atc act tat cac 4056 Met Arg Leu Leu Ala Asn Tyr Ala Ser Gln Asn Ile Thr Tyr His 1280 1285 1290 tgc aaa aat tcc att gca tat atg gat gag gag aca gga aat ttg 4101 Cys Lys Asn Ser Ile Ala Tyr Met Asp Glu Glu Thr Gly Asn Leu 1295 1300 1305 aag aaa gca gtt att ctc caa ggt agt aac gat gtt gag ctt gtg 4146 Lys Lys Ala Val Ile Leu Gln Gly Ser Asn Asp Val Glu Leu Val 1310 1315 1320 gct gag gga aat agt aga ttc act tac aca gtt ttg gtg gat gga 4191 Ala Glu Gly Asn Ser Arg Phe Thr Tyr Thr Val Leu Val Asp Gly 1325 1330 1335 tgc tca aag aaa act aat gag tgg ggc aag aca atc att gag tac 4236 Cys Ser Lys Lys Thr Asn Glu Trp Gly Lys Thr Ile Ile Glu Tyr 1340 1345 1350 aag aca aat aag cct tct agg ctc cca ttt ctc gat att gca cct 4281 Lys Thr Asn Lys Pro Ser Arg Leu Pro Phe Leu Asp Ile Ala Pro 1355 1360 1365 ctt gat atc gga gga gct gat cac gag ttt ttt gtt gat atc gga 4326 Leu Asp Ile Gly Gly Ala Asp His Glu Phe Phe Val Asp Ile Gly 1370 1375 1380 cct gtt tgt ttt aag taa tgagctcgcg gccgcatc 4362 Pro Val Cys Phe Lys 1385 <210> 6 <211> 1389 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Leu 35 40 45 Gln Glu Glu Thr Val Arg Lys Gly Pro Ala Gly Asp Arg Gly Pro Arg 50 55 60 Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Arg Asp Gly Glu Asp Gly 65 70 75 80 Pro Thr Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu 85 90 95 Gly Gly Asn Phe Ala Ala Gln Tyr Asp Gly Lys Gly Val Gly Leu Gly 100 105 110 Pro Gly Pro Met Gly Leu Met Gly Pro Arg Gly Pro Pro Gly Ala Ala 115 120 125 Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Ala Gly Glu Pro Gly 130 135 140 Glu Pro Gly Gln Thr Gly Pro Ala Gly Ala Arg Gly Pro Ala Gly Pro 145 150 155 160 Pro Gly Lys Ala Gly Glu Asp Gly His Pro Gly Lys Pro Gly Arg Pro 165 170 175 Gly Glu Arg Gly Val Val Gly Pro Gln Gly Ala Arg Gly Phe Pro Gly 180 185 190 Thr Pro Gly Leu Pro Gly Phe Lys Gly Ile Arg Gly His Asn Gly Leu 195 200 205 Asp Gly Leu Lys Gly Gln Pro Gly Ala Pro Gly Val Lys Gly Glu Pro 210 215 220 Gly Ala Pro Gly Glu Asn Gly Thr Pro Gly Gln Thr Gly Ala Arg Gly 225 230 235 240 Leu Pro Gly Glu Arg Gly Arg Val Gly Ala Pro Gly Pro Ala Gly Ala 245 250 255 Arg Gly Ser Asp Gly Ser Val Gly Pro Val Gly Pro Ala Gly Pro Ile 260 265 270 Gly Ser Ala Gly Pro Pro Gly Phe Pro Gly Ala Pro Gly Pro Lys Gly 275 280 285 Glu Ile Gly Ala Val Gly Asn Ala Gly Pro Thr Gly Pro Ala Gly Pro 290 295 300 Arg Gly Glu Val Gly Leu Pro Gly Leu Ser Gly Pro Val Gly Pro Pro 305 310 315 320 Gly Asn Pro Gly Ala Asn Gly Leu Thr Gly Ala Lys Gly Ala Ala Gly 325 330 335 Leu Pro Gly Val Ala Gly Ala Pro Gly Leu Pro Gly Pro Arg Gly Ile 340 345 350 Pro Gly Pro Val Gly Ala Ala Gly Ala Thr Gly Ala Arg Gly Leu Val 355 360 365 Gly Glu Pro Gly Pro Ala Gly Ser Lys Gly Glu Ser Gly Asn Lys Gly 370 375 380 Glu Pro Gly Ser Ala Gly Pro Gln Gly Pro Pro Gly Pro Ser Gly Glu 385 390 395 400 Glu Gly Lys Arg Gly Pro Asn Gly Glu Ala Gly Ser Ala Gly Pro Pro 405 410 415 Gly Pro Pro Gly Leu Arg Gly Ser Pro Gly Ser Arg Gly Leu Pro Gly 420 425 430 Ala Asp Gly Arg Ala Gly Val Met Gly Pro Pro Gly Ser Arg Gly Ala 435 440 445 Ser Gly Pro Ala Gly Val Arg Gly Pro Asn Gly Asp Ala Gly Arg Pro 450 455 460 Gly Glu Pro Gly Leu Met Gly Pro Arg Gly Leu Pro Gly Ser Pro Gly 465 470 475 480 Asn Ile Gly Pro Ala Gly Lys Glu Gly Pro Val Gly Leu Pro Gly Ile 485 490 495 Asp Gly Arg Pro Gly Pro Ile Gly Pro Ala Gly Ala Arg Gly Glu Pro 500 505 510 Gly Asn Ile Gly Phe Pro Gly Pro Lys Gly Pro Thr Gly Asp Pro Gly 515 520 525 Lys Asn Gly Asp Lys Gly His Ala Gly Leu Ala Gly Ala Arg Gly Ala 530 535 540 Pro Gly Pro Asp Gly Asn Asn Gly Ala Gln Gly Pro Pro Gly Pro Gln 545 550 555 560 Gly Val Gln Gly Gly Lys Gly Glu Gln Gly Pro Ala Gly Pro Pro Gly 565 570 575 Phe Gln Gly Leu Pro Gly Pro Ser Gly Pro Ala Gly Glu Val Gly Lys 580 585 590 Pro Gly Glu Arg Gly Leu His Gly Glu Phe Gly Leu Pro Gly Pro Ala 595 600 605 Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly Glu Ser Gly Ala Ala Gly 610 615 620 Pro Thr Gly Pro Ile Gly Ser Arg Gly Pro Ser Gly Pro Pro Gly Pro 625 630 635 640 Asp Gly Asn Lys Gly Glu Pro Gly Val Val Gly Ala Val Gly Thr Ala 645 650 655 Gly Pro Ser Gly Pro Ser Gly Leu Pro Gly Glu Arg Gly Ala Ala Gly 660 665 670 Ile Pro Gly Gly Lys Gly Glu Lys Gly Glu Pro Gly Leu Arg Gly Glu 675 680 685 Ile Gly Asn Pro Gly Arg Asp Gly Ala Arg Gly Ala His Gly Ala Val 690 695 700 Gly Ala Pro Gly Pro Ala Gly Ala Thr Gly Asp Arg Gly Glu Ala Gly 705 710 715 720 Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Ser Pro Gly Glu 725 730 735 Arg Gly Glu Val Gly Pro Ala Gly Pro Asn Gly Phe Ala Gly Pro Ala 740 745 750 Gly Ala Ala Gly Gln Pro Gly Ala Lys Gly Glu Arg Gly Gly Lys Gly 755 760 765 Pro Lys Gly Glu Asn Gly Val Val Gly Pro Thr Gly Pro Val Gly Ala 770 775 780 Ala Gly Pro Ala Gly Pro Asn Gly Pro Pro Gly Pro Ala Gly Ser Arg 785 790 795 800 Gly Asp Gly Gly Pro Pro Gly Met Thr Gly Phe Pro Gly Ala Ala Gly 805 810 815 Arg Thr Gly Pro Pro Gly Pro Ser Gly Ile Ser Gly Pro Pro Gly Pro 820 825 830 Pro Gly Pro Ala Gly Lys Glu Gly Leu Arg Gly Pro Arg Gly Asp Gln 835 840 845 Gly Pro Val Gly Arg Thr Gly Glu Val Gly Ala Val Gly Pro Pro Gly 850 855 860 Phe Ala Gly Glu Lys Gly Pro Ser Gly Glu Ala Gly Thr Ala Gly Pro 865 870 875 880 Pro Gly Thr Pro Gly Pro Gln Gly Leu Leu Gly Ala Pro Gly Ile Leu 885 890 895 Gly Leu Pro Gly Ser Arg Gly Glu Arg Gly Leu Pro Gly Val Ala Gly 900 905 910 Ala Val Gly Glu Pro Gly Pro Leu Gly Ile Ala Gly Pro Pro Gly Ala 915 920 925 Arg Gly Pro Pro Gly Ala Val Gly Ser Pro Gly Val Asn Gly Ala Pro 930 935 940 Gly Glu Ala Gly Arg Asp Gly Asn Pro Gly Asn Asp Gly Pro Pro Gly 945 950 955 960 Arg Asp Gly Gln Pro Gly His Lys Gly Glu Arg Gly Tyr Pro Gly Asn 965 970 975 Ile Gly Pro Val Gly Ala Ala Gly Ala Pro Gly Pro His Gly Pro Val 980 985 990 Gly Pro Ala Gly Lys His Gly Asn Arg Gly Glu Thr Gly Pro Ser Gly 995 1000 1005 Pro Val Gly Pro Ala Gly Ala Val Gly Pro Arg Gly Pro Ser Gly 1010 1015 1020 Pro Gln Gly Ile Arg Gly Asp Lys Gly Glu Pro Gly Glu Lys Gly 1025 1030 1035 Pro Arg Gly Leu Pro Gly Phe Lys Gly His Asn Gly Leu Gln Gly 1040 1045 1050 Leu Pro Gly Ile Ala Gly His His Gly Asp Gln Gly Ala Pro Gly 1055 1060 1065 Ser Val Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly Pro Ser Gly 1070 1075 1080 Pro Ala Gly Lys Asp Gly Arg Thr Gly His Pro Gly Thr Val Gly 1085 1090 1095 Pro Ala Gly Ile Arg Gly Pro Gln Gly His Gln Gly Pro Ala Gly 1100 1105 1110 Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Val Ser Gly 1115 1120 1125 Gly Gly Tyr Asp Phe Gly Tyr Asp Gly Asp Phe Tyr Arg Ala Asp 1130 1135 1140 Gln Pro Arg Ser Ala Pro Ser Leu Arg Pro Lys Asp Tyr Glu Val 1145 1150 1155 Asp Ala Thr Leu Lys Ser Leu Asn Asn Gln Ile Glu Thr Leu Leu 1160 1165 1170 Thr Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp 1175 1180 1185 Leu Arg Leu Ser His Pro Glu Trp Ser Ser Gly Tyr Tyr Trp Ile 1190 1195 1200 Asp Pro Asn Gln Gly Cys Thr Met Glu Ala Ile Lys Val Tyr Cys 1205 1210 1215 Asp Phe Pro Thr Gly Glu Thr Cys Ile Arg Ala Gln Pro Glu Asn 1220 1225 1230 Ile Pro Ala Lys Asn Trp Tyr Arg Ser Ser Lys Asp Lys Lys His 1235 1240 1245 Val Trp Leu Gly Glu Thr Ile Asn Ala Gly Ser Gln Phe Glu Tyr 1250 1255 1260 Asn Val Glu Gly Val Thr Ser Lys Glu Met Ala Thr Gln Leu Ala 1265 1270 1275 Phe Met Arg Leu Leu Ala Asn Tyr Ala Ser Gln Asn Ile Thr Tyr 1280 1285 1290 His Cys Lys Asn Ser Ile Ala Tyr Met Asp Glu Glu Thr Gly Asn 1295 1300 1305 Leu Lys Lys Ala Val Ile Leu Gln Gly Ser Asn Asp Val Glu Leu 1310 1315 1320 Val Ala Glu Gly Asn Ser Arg Phe Thr Tyr Thr Val Leu Val Asp 1325 1330 1335 Gly Cys Ser Lys Lys Thr Asn Glu Trp Gly Lys Thr Ile Ile Glu 1340 1345 1350 Tyr Lys Thr Asn Lys Pro Ser Arg Leu Pro Phe Leu Asp Ile Ala 1355 1360 1365 Pro Leu Asp Ile Gly Gly Ala Asp His Glu Phe Phe Val Asp Ile 1370 1375 1380 Gly Pro Val Cys Phe Lys 1385 <210> 7 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding region of the appoplast signal of Arabidopsis thaliana endo-1,4-beta-glucanase and flanking regions <400> 7 gccatggcta ggaagtcttt gattttccca gtgattcttc ttgctgtgct tcttttctct 60 ccacctattt actctgctgg acacgattat agggatgctc ttaggaagtc atctatggct 120 caattgc 127 <210> 8 <211> 127 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence of the appoplast signal of Arabidopsis thaliana endo-1,4-beta-glucanase and flanking regions <220> <221> CDS <222> (10)..(120) <400> 8 gccatggct agg aag tct ttg att ttc cca gtg att ctt ctt gct gtg ctt 51 Arg Lys Ser Leu Ile Phe Pro Val Ile Leu Leu Ala Val Leu 1 5 10 ctt ttc tct cca cct att tac tct gct gga cac gat tat agg gat gct 99 Leu Phe Ser Pro Pro Ile Tyr Ser Ala Gly His Asp Tyr Arg Asp Ala 15 20 25 30 ctt agg aag tca tct atg gct caattgc 127 Leu Arg Lys Ser Ser Met Ala 35 <210> 9 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 9 Arg Lys Ser Leu Ile Phe Pro Val Ile Leu Leu Ala Val Leu Leu Phe 1 5 10 15 Ser Pro Pro Ile Tyr Ser Ala Gly His Asp Tyr Arg Asp Ala Leu Arg 20 25 30 Lys Ser Ser Met Ala 35 <210> 10 <211> 1037 <212> DNA <213> Artificial Sequence <220> <223> Chrysanthemum rbcS1 promoter and 5'UTR <400> 10 aaatggcgcg ccaagcttag acaaacaccc cttgttatac aaagaatttc gctttacaaa 60 atcaaattcg agaaaataat atatgcacta aataagatca ttcggatcca atctaaccaa 120 ttacgatacg ctttgggtac acttgatttt tgtttcagta gttacatata tcttgtttta 180 tatgctatct ttaaggatct tcactcaaag actatttgtt gatgttcttg atggggctcg 240 gaagatttga tatgatacac tctaatcttt aggagatacc agccaggatt atattcagta 300 agacaatcaa attttacgtg ttcaaactcg ttatcttttc atttaatgga tgagccagaa 360 tctctataga atgattgcaa tcgagaatat gttcggccga tatccctttg ttggcttcaa 420 tattctacat atcacacaag aatcgaccgt attgtaccct ctttccataa aggaacacac 480 agtatgcaga tgcttttttc ccacatgcag taacataggt attcaaaaat ggctaaaaga 540 agttggataa caaattgaca actatttcca tttctgttat ataaatttca caacacacaa 600 aagcccgtaa tcaagagtct gcccatgtac gaaataactt ctattatttg gtattgggcc 660 taagcccagc tcagagtacg tgggggtacc acatatagga aggtaacaaa atactgcaag 720 atagccccat aacgtaccag cctctcctta ccacgaagag ataagatata agacccaccc 780 tgccacgtgt cacatcgtca tggtggttaa tgataaggga ttacatcctt ctatgtttgt 840 ggacatgatg catgtaatgt catgagccac atgatccaat ggccacagga acgtaagaat 900 gtagatagat ttgattttgt ccgttagata gcaaacaaca ttataaaagg tgtgtatcaa 960 tacgaactaa ttcactcatt ggattcatag aagtccattc ctcctaagta tctaaacata 1020 tgcaattgtc gactaaa 1037 <210> 11 <211> 975 <212> DNA <213> Artificial Sequence <220> <223> Chrysanthemum rbcS1 3'UTR and terminator <400> 11 aaaaggatcc gcggccgcat aagttttact atttaccaag acttttgaat attaaccttc 60 ttgtaacgag tcggttaaat ttgattgttt agggttttgt attatttttt tttggtcttt 120 taattcatca ctttaattcc ctaattgtct gttcatttcg ttgtttgttt ccggatcgat 180 aatgaaatgt aagagatatc atatataaat aataaattgt cgtttcatat ttgcaatctt 240 tttttacaaa cctttaatta attgtatgta tgacattttc ttcttgttat attaggggga 300 aataatgtta aataaaagta caaaataaac tacagtacat cgtactgaat aaattaccta 360 gccaaaaagt acacctttcc atatacttcc tacatgaagg cattttcaac attttcaaat 420 aaggaatgct acaaccgcat aataacatcc acaaattttt ttataaaata acatgtcaga 480 cagtgattga aagattttat tatagtttcg ttatcttctt ttctcattaa gcgaatcact 540 acctaacacg tcattttgtg aaatattttt tgaatgtttt tatatagttg tagcattcct 600 cttttcaaat tagggtttgt ttgagatagc atttcagccg gttcatacaa cttaaaagca 660 tactctaatg ctggaaaaaa gactaaaaaa tcttgtaagt tagcgcagaa tattgaccca 720 aattatatac acacatgacc ccatatagag actaattaca cttttaacca ctaataatta 780 ttactgtatt ataacatcta ctaattaaac ttgtgagttt ttgctagaat tattatcata 840 tatactaaaa ggcaggaacg caaacattgc cccggtactg tagcaactac ggtagacgca 900 ttaattgtct atagtggacg cattaattaa ccaaaaccgc ctctttcccc ttcttcttga 960 agcttgagct ctttt 975 <210> 12 <211> 1633 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Prolyl 4-hydroxylase beta subunit and flanking regions <400> 12 ctcgagtaaa ccatggctca tgctagggtt ttgcttttgg ctcttgctgt tcttgctact 60 gctgctgttg ctgtggcttc ttcttcatct ttcgctgatt ctaacccaat taggccagtg 120 actgatagag ctgcttctac tcttgctcaa ttggtcgaca tggatgctcc agaagaggag 180 gatcacgttc ttgtgcttag gaagtctaac ttcgctgaag ctcttgctgc tcacaagtac 240 cttcttgtgg agttttatgc tccttggtgc ggacattgca aagctcttgc tccagagtat 300 gctaaggctg ctggaaagtt gaaggctgag ggatctgaaa ttaggcttgc taaagtggat 360 gctactgagg agtctgatct tgctcaacag tacggagtta ggggataccc aactattaag 420 ttcttcagga acggagatac tgcttctcca aaggagtata ctgctggaag ggaggctgat 480 gatattgtga actggcttaa gaagagaact ggaccagctg ctactactct tccagatgga 540 gctgctgctg aatctcttgt ggagtcatct gaggtggcag tgattggatt cttcaaggat 600 gtggagtctg attctgctaa gcagttcctt caagctgctg aggctattga tgatattcca 660 ttcggaatta cttctaactc tgatgtgttc tctaagtacc agcttgataa ggatggagtg 720 gtgcttttca agaaattcga tgagggaagg aacaatttcg agggagaggt gacaaaggag 780 aaccttcttg atttcattaa gcacaaccag cttccacttg tgattgagtt cactgagcag 840 actgctccaa agattttcgg aggagagatt aagactcaca ttcttctttt ccttccaaag 900 tctgtgtctg attacgatgg aaagttgtct aacttcaaga ctgctgctga gtctttcaag 960 ggaaagattc ttttcatttt cattgattct gatcacactg ataaccagag gattcttgag 1020 ttcttcggac ttaagaagga agagtgccca gctgttaggc ttattactct tgaggaggag 1080 atgactaagt acaagccaga gtctgaagaa cttactgctg agaggattac tgagttctgc 1140 cacagattcc ttgagggaaa gattaagcca caccttatgt ctcaagagct tccagaggat 1200 tgggataagc agccagttaa ggtgttggtg ggtaaaaact tcgaggatgt ggctttcgat 1260 gagaagaaga acgtgttcgt ggagttctac gcaccttggt gtggtcactg taagcagctt 1320 gctccaattt gggataagtt gggagagact tacaaggatc acgagaacat tgtgattgct 1380 aagatggatt ctactgctaa cgaggtggag gctgttaagg ttcactcttt cccaactttg 1440 aagttcttcc cagcttctgc tgataggact gtgattgatt acaacggaga aaggactctt 1500 gatggattca agaagttcct tgagtctgga ggacaagatg gagctggaga tgatgatgat 1560 cttgaggatt tggaagaagc tgaggagcca gatatggagg aggatgatga tcagaaggct 1620 gtgtgatgag ctc 1633 <210> 13 <211> 537 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence containing the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Prolyl 4-hydroxylase beta subunit and flanking regions <400> 13 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Val 35 40 45 Asp Met Asp Ala Pro Glu Glu Glu Asp His Val Leu Val Leu Arg Lys 50 55 60 Ser Asn Phe Ala Glu Ala Leu Ala Ala His Lys Tyr Leu Leu Val Glu 65 70 75 80 Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Pro Glu Tyr 85 90 95 Ala Lys Ala Ala Gly Lys Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu 100 105 110 Ala Lys Val Asp Ala Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly 115 120 125 Val Arg Gly Tyr Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala 130 135 140 Ser Pro Lys Glu Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn 145 150 155 160 Trp Leu Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly 165 170 175 Ala Ala Ala Glu Ser Leu Val Glu Ser Ser Glu Val Ala Val Ile Gly 180 185 190 Phe Phe Lys Asp Val Glu Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala 195 200 205 Ala Glu Ala Ile Asp Asp Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp 210 215 220 Val Phe Ser Lys Tyr Gln Leu Asp Lys Asp Gly Val Val Leu Phe Lys 225 230 235 240 Lys Phe Asp Glu Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu 245 250 255 Asn Leu Leu Asp Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu 260 265 270 Phe Thr Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr 275 280 285 His Ile Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys 290 295 300 Leu Ser Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu 305 310 315 320 Phe Ile Phe Ile Asp Ser Asp His Thr Asp Asn Gln Arg Ile Leu Glu 325 330 335 Phe Phe Gly Leu Lys Lys Glu Glu Cys Pro Ala Val Arg Leu Ile Thr 340 345 350 Leu Glu Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr 355 360 365 Ala Glu Arg Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile 370 375 380 Lys Pro His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln 385 390 395 400 Pro Val Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp 405 410 415 Glu Lys Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His 420 425 430 Cys Lys Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys 435 440 445 Asp His Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu 450 455 460 Val Glu Ala Val Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro 465 470 475 480 Ala Ser Ala Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu 485 490 495 Asp Gly Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly 500 505 510 Asp Asp Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met 515 520 525 Glu Glu Asp Asp Asp Gln Lys Ala Val 530 535 <210> 14 <211> 1723 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Prolyl 4-hydroxylase alpha-1 subunit and flanking regions <400> 14 ctcgagtaaa ccatggctca tgctagggtt ttgcttttgg ctcttgctgt tcttgctact 60 gctgctgttg ctgtggcttc ttcttcatct ttcgctgatt ctaacccaat taggccagtg 120 actgatagag ctgcttctac tcttgctcaa ttggtcgaca tgcacccagg attcttcact 180 tctattggac agatgactga tcttattcac actgagaagg atcttgtgac ttctcttaag 240 gattacatta aggctgagga ggataagttg gagcagatta agaagtgggc tgagaagttg 300 gataggctta cttctactgc tacaaaagat ccagagggat tcgttggtca tccagtgaac 360 gctttcaagt tgatgaagag gcttaacact gagtggagtg agcttgagaa ccttgtgctt 420 aaggatatgt ctgatggatt catttctaac cttactattc agaggcagta cttcccaaat 480 gatgaggatc aagtgggagc tgctaaggct cttcttaggc ttcaggatac ttacaacctt 540 gatactgata caatttctaa gggaaacctt ccaggagtta agcacaagtc tttccttact 600 gctgaggatt gcttcgagct tggaaaggtt gcatacactg aggctgatta ctaccacact 660 gagctttgga tggaacaagc tcttaggcaa cttgatgagg gagagatttc tactattgat 720 aaggtgtcag tgcttgatta cctttcttac gctgtgtacc agcagggtga tcttgataag 780 gctcttttgc ttactaagaa gttgcttgag cttgatccag aacatcagag ggctaacgga 840 aaccttaagt acttcgagta cattatggct aaggaaaagg atgtgaacaa gtctgcttct 900 gatgatcagt ctgatcaaaa gactactcca aagaagaagg gagtggctgt tgattatctt 960 cctgagaggc agaagtatga gatgttgtgt aggggagagg gtattaagat gactccaagg 1020 aggcagaaga agttgttctg caggtatcac gatggaaaca ggaacccaaa gttcattctt 1080 gctccagcta agcaagaaga tgagtgggat aagccaagga ttattaggtt ccacgatatt 1140 atttctgatg ctgagattga gattgtgaag gatcttgcta agccaagact taggagggct 1200 actatttcta accctattac tggtgatctt gagactgtgc actacaggat ttctaagtct 1260 gcttggcttt ctggatacga gaacccagtg gtgtctagga ttaacatgag gattcaggat 1320 cttactggac ttgatgtgtc tactgctgag gagcttcaag ttgctaacta cggagttgga 1380 ggacaatatg agccacactt cgatttcgct aggaaggatg agccagatgc ttttaaggag 1440 cttggaactg gaaacaggat tgctacttgg cttttctaca tgtctgatgt ttctgctgga 1500 ggagctactg ttttcccaga agtgggagct tctgtttggc caaagaaggg aactgctgtg 1560 ttctggtaca accttttcgc ttctggagag ggagattact ctactaggca tgctgcttgc 1620 ccagttcttg ttggaaacaa gtgggtgtca aacaagtggc ttcatgagag gggacaagag 1680 tttagaaggc catgcactct ttctgagctt gagtgatgag ctc 1723 <210> 15 <211> 567 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence containing the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Prolyl 4-hydroxylase alpha-1 subunit and flanking regions <400> 15 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Val 35 40 45 Asp Met His Pro Gly Phe Phe Thr Ser Ile Gly Gln Met Thr Asp Leu 50 55 60 Ile His Thr Glu Lys Asp Leu Val Thr Ser Leu Lys Asp Tyr Ile Lys 65 70 75 80 Ala Glu Glu Asp Lys Leu Glu Gln Ile Lys Lys Trp Ala Glu Lys Leu 85 90 95 Asp Arg Leu Thr Ser Thr Ala Thr Lys Asp Pro Glu Gly Phe Val Gly 100 105 110 His Pro Val Asn Ala Phe Lys Leu Met Lys Arg Leu Asn Thr Glu Trp 115 120 125 Ser Glu Leu Glu Asn Leu Val Leu Lys Asp Met Ser Asp Gly Phe Ile 130 135 140 Ser Asn Leu Thr Ile Gln Arg Gln Tyr Phe Pro Asn Asp Glu Asp Gln 145 150 155 160 Val Gly Ala Ala Lys Ala Leu Leu Arg Leu Gln Asp Thr Tyr Asn Leu 165 170 175 Asp Thr Asp Thr Ile Ser Lys Gly Asn Leu Pro Gly Val Lys His Lys 180 185 190 Ser Phe Leu Thr Ala Glu Asp Cys Phe Glu Leu Gly Lys Val Ala Tyr 195 200 205 Thr Glu Ala Asp Tyr Tyr His Thr Glu Leu Trp Met Glu Gln Ala Leu 210 215 220 Arg Gln Leu Asp Glu Gly Glu Ile Ser Thr Ile Asp Lys Val Ser Val 225 230 235 240 Leu Asp Tyr Leu Ser Tyr Ala Val Tyr Gln Gln Gly Asp Leu Asp Lys 245 250 255 Ala Leu Leu Leu Thr Lys Lys Leu Leu Glu Leu Asp Pro Glu His Gln 260 265 270 Arg Ala Asn Gly Asn Leu Lys Tyr Phe Glu Tyr Ile Met Ala Lys Glu 275 280 285 Lys Asp Val Asn Lys Ser Ala Ser Asp Asp Gln Ser Asp Gln Lys Thr 290 295 300 Thr Pro Lys Lys Lys Gly Val Ala Val Asp Tyr Leu Pro Glu Arg Gln 305 310 315 320 Lys Tyr Glu Met Leu Cys Arg Gly Glu Gly Ile Lys Met Thr Pro Arg 325 330 335 Arg Gln Lys Lys Leu Phe Cys Arg Tyr His Asp Gly Asn Arg Asn Pro 340 345 350 Lys Phe Ile Leu Ala Pro Ala Lys Gln Glu Asp Glu Trp Asp Lys Pro 355 360 365 Arg Ile Ile Arg Phe His Asp Ile Ile Ser Asp Ala Glu Ile Glu Ile 370 375 380 Val Lys Asp Leu Ala Lys Pro Arg Leu Arg Arg Ala Thr Ile Ser Asn 385 390 395 400 Pro Ile Thr Gly Asp Leu Glu Thr Val His Tyr Arg Ile Ser Lys Ser 405 410 415 Ala Trp Leu Ser Gly Tyr Glu Asn Pro Val Val Ser Arg Ile Asn Met 420 425 430 Arg Ile Gln Asp Leu Thr Gly Leu Asp Val Ser Thr Ala Glu Glu Leu 435 440 445 Gln Val Ala Asn Tyr Gly Val Gly Gly Gln Tyr Glu Pro His Phe Asp 450 455 460 Phe Ala Arg Lys Asp Glu Pro Asp Ala Phe Lys Glu Leu Gly Thr Gly 465 470 475 480 Asn Arg Ile Ala Thr Trp Leu Phe Tyr Met Ser Asp Val Ser Ala Gly 485 490 495 Gly Ala Thr Val Phe Pro Glu Val Gly Ala Ser Val Trp Pro Lys Lys 500 505 510 Gly Thr Ala Val Phe Trp Tyr Asn Leu Phe Ala Ser Gly Glu Gly Asp 515 520 525 Tyr Ser Thr Arg His Ala Ala Cys Pro Val Leu Val Gly Asn Lys Trp 530 535 540 Val Ser Asn Lys Trp Leu His Glu Arg Gly Gln Glu Phe Arg Arg Pro 545 550 555 560 Cys Thr Leu Ser Glu Leu Glu 565 <210> 16 <211> 928 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the plant Prolyl 4-hydroxylase Plant and flanking regions <400> 16 ctcgagtaaa ccatggctca tgctagggtt ttgcttttgg ctcttgctgt tcttgctact 60 gctgctgttg ctgtggcttc ttcttcatct ttcgctgatt ctaacccaat taggccagtg 120 actgatagag ctgcttctac tcttgctcaa ttggtcgaca tgcttggtat tctttctctt 180 ccaaacgcta acaggaactc ttctaagact aacgatctta ctaacattgt gaggaagtct 240 gagacttctt ctggagatga ggagggaaat ggagaaagat gggtggaagt gatttcttgg 300 gagccaaggg ctgttgttta ccacaacttc cttactaatg aggagtgcga gcaccttatt 360 tctcttgcta agccatctat ggtgaagtct actgtggtgg atgagaaaac tggaggatct 420 aaggattcaa gagtgaggac ttcatctggt actttcctta ggaggggaca tgatgaagtt 480 gtggaagtta ttgagaagag gatttctgat ttcactttca ttccagtgga gaacggagaa 540 ggacttcaag ttcttcacta ccaagtggga caaaagtacg agccacacta cgattacttc 600 cttgatgagt tcaacactaa gaacggagga cagaggattg ctactgtgct tatgtacctt 660 tctgatgtgg atgatggagg agagactgtt tttccagctg ctaggggaaa catttctgct 720 gttccttggt ggaacgagct ttctaagtgt ggaaaggagg gactttctgt gcttccaaag 780 aaaagggatg ctcttctttt ctggaacatg aggccagatg cttctcttga tccatcttct 840 cttcatggag gatgcccagt tgttaaggga aacaagtggt catctactaa gtggttccac 900 gtgcacgagt tcaaggtgta atgagctc 928 <210> 17 <211> 302 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence containing the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the plant Prolyl 4-hydroxylase Plant and flanking regions <400> 17 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Val 35 40 45 Asp Met Leu Gly Ile Leu Ser Leu Pro Asn Ala Asn Arg Asn Ser Ser 50 55 60 Lys Thr Asn Asp Leu Thr Asn Ile Val Arg Lys Ser Glu Thr Ser Ser 65 70 75 80 Gly Asp Glu Glu Gly Asn Gly Glu Arg Trp Val Glu Val Ile Ser Trp 85 90 95 Glu Pro Arg Ala Val Val Tyr His Asn Phe Leu Thr Asn Glu Glu Cys 100 105 110 Glu His Leu Ile Ser Leu Ala Lys Pro Ser Met Val Lys Ser Thr Val 115 120 125 Val Asp Glu Lys Thr Gly Gly Ser Lys Asp Ser Arg Val Arg Thr Ser 130 135 140 Ser Gly Thr Phe Leu Arg Arg Gly His Asp Glu Val Val Glu Val Ile 145 150 155 160 Glu Lys Arg Ile Ser Asp Phe Thr Phe Ile Pro Val Glu Asn Gly Glu 165 170 175 Gly Leu Gln Val Leu His Tyr Gln Val Gly Gln Lys Tyr Glu Pro His 180 185 190 Tyr Asp Tyr Phe Leu Asp Glu Phe Asn Thr Lys Asn Gly Gly Gln Arg 195 200 205 Ile Ala Thr Val Leu Met Tyr Leu Ser Asp Val Asp Asp Gly Gly Glu 210 215 220 Thr Val Phe Pro Ala Ala Arg Gly Asn Ile Ser Ala Val Pro Trp Trp 225 230 235 240 Asn Glu Leu Ser Lys Cys Gly Lys Glu Gly Leu Ser Val Leu Pro Lys 245 250 255 Lys Arg Asp Ala Leu Leu Phe Trp Asn Met Arg Pro Asp Ala Ser Leu 260 265 270 Asp Pro Ser Ser Leu His Gly Gly Cys Pro Val Val Lys Gly Asn Lys 275 280 285 Trp Ser Ser Thr Lys Trp Phe His Val His Glu Phe Lys Val 290 295 300 <210> 18 <211> 2689 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the human Procollagen C-proteinase and flanking regions <400> 18 agatctatcg atgcatgcca tggtaccgcg ccatggctca attggctgca acatcaaggc 60 ctgaaagagt ttggccagat ggtgttattc ctttcgttat tggtggaaac tttactggat 120 ctcagagagc agtttttaga caagctatga gacattggga aaagcacact tgtgtgacat 180 tccttgaaag gactgatgaa gattcttata ttgtgttcac ataccgtcca tgtggatgct 240 gctcatatgt tggtagaagg ggaggaggtc cacaagcaat ttctattgga aaaaactgcg 300 ataagttcgg aattgtggtg catgaattgg gacatgttgt tggtttctgg cacgaacaca 360 caaggccaga tagggatagg cacgtgtcta ttgtgaggga aaacattcag ccaggtcaag 420 agtacaattt tcttaagatg gaacctcaag aggtggaatc tctcggagag acttacgact 480 tcgactccat catgcactac gcaaggaata ctttcagcag gggcatcttc ttggatacca 540 ttgtgcctaa gtacgaggtg aacggcgtta agccacctat tggtcaaagg actaggctct 600 ctaagggtga tattgcacag gctaggaagc tctacaaatg tccagcatgc ggagaaactc 660 ttcaggattc cactggcaac ttctcatctc cagagtaccc aaacggatac tctgctcata 720 tgcactgtgt ttggaggatc tcagtgactc ctggagagaa gatcatcctc aacttcactt 780 ccctcgatct ctatcgttct aggctctgtt ggtacgacta tgtggaagtg agagatggct 840 tctggagaaa ggctccactt agaggaaggt tctgcggatc taaacttcct gagccaatcg 900 tgtctactga ttccagattg tgggtggagt tcaggtcctc ttctaattgg gttggcaagg 960 gcttttttgc tgtgtacgag gctatttgtg gcggcgacgt gaaaaaggac tacggacata 1020 ttcaaagtcc aaattaccca gatgattacc gtccttcaaa agtgtgtatt tggaggattc 1080 aagtgagtga gggtttccat gttggattga cattccaatc tttcgaaatt gagagacacg 1140 attcatgcgc atacgattat ttggaagtga gagatggaca ctctgaatct tctacactta 1200 ttggaaggta ctgcggttat gagaaacctg atgatattaa gtctacttct agtaggttgt 1260 ggcttaaatt tgtgtcagat ggttctatta acaaggctgg tttcgcagtg aacttcttca 1320 aggaagtgga tgaatgctca agacctaaca gaggaggatg tgagcaaaga tgccttaaca 1380 ctttgggaag ttacaagtgt tcttgcgatc ctggatacga gttggctcct gataagagaa 1440 gatgcgaagc tgcttgcggt ggttttttga caaaattgaa cggatctatt acttctcctg 1500 gatggccaaa agagtaccca cctaataaga attgcatttg gcagcttgtt gcacctactc 1560 agtaccgtat ttcattgcaa ttcgattttt tcgagactga gggtaatgat gtgtgcaagt 1620 acgatttcgt ggaagtgaga tcaggtctta ctgctgatag taaattgcac ggaaagttct 1680 gcggatctga aaaaccagaa gtgattacat cacagtacaa caatatgagg gtggagttca 1740 aatctgataa tactgtttct aaaaaaggtt ttaaggcaca tttcttttct gataaggacg 1800 agtgctctaa agataatggt ggttgccagc aggattgcgt gaacacattc ggttcatatg 1860 agtgccaatg ccgtagtgga tttgttcttc acgataacaa acatgattgc aaagaggcag 1920 gttgcgatca caaggtgaca tctacttcag gtactatcac atctccaaac tggcctgata 1980 agtatccttc aaaaaaagaa tgtacatggg caatttcttc tacaccaggt catagggtta 2040 agttgacatt catggagatg gatattgaga gtcaaccaga gtgcgcttat gatcatcttg 2100 aggtgttcga tggaagggat gctaaggctc ctgttcttgg tagattctgt ggtagtaaaa 2160 agccagaacc agtgcttgca acaggatcta ggatgttcct tagattctac tctgataact 2220 cagttcagag gaaaggattc caagctagtc acgcaactga atgcggtgga caagttagag 2280 cagatgttaa gactaaggat ctttactcac acgcacagtt cggagataac aactaccctg 2340 gaggagttga ttgcgagtgg gttattgtgg ctgaagaggg atacggagtt gagcttgttt 2400 tccagacatt cgaggtggag gaggaaactg attgcggtta cgattatatg gaactttttg 2460 atggatacga tagtactgct ccaagacttg gaaggtattg tggtagtggt ccaccagaag 2520 aggtgtactc agctggagat agtgttcttg ttaagttcca cagtgatgat acaattacta 2580 agaagggatt ccatcttaga tatacttcaa ctaagtttca ggatactctt cattctagga 2640 agtaatgagc tcgcggccgc atccaagctt ctgcagacgc gtcgacgtc 2689 <210> 19 <211> 870 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence containing the human Procollagen C-proteinase and flanking regions <400> 19 Met Ala Gln Leu Ala Ala Thr Ser Arg Pro Glu Arg Val Trp Pro Asp 1 5 10 15 Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr Gly Ser Gln Arg 20 25 30 Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys His Thr Cys Val 35 40 45 Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile Val Phe Thr Tyr 50 55 60 Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg Gly Gly Gly Pro 65 70 75 80 Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe Gly Ile Val Val 85 90 95 His Glu Leu Gly His Val Val Gly Phe Trp His Glu His Thr Arg Pro 100 105 110 Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn Ile Gln Pro Gly 115 120 125 Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu Val Glu Ser Leu 130 135 140 Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His Tyr Ala Arg Asn Thr 145 150 155 160 Phe Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro Lys Tyr Glu Val 165 170 175 Asn Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg Leu Ser Lys Gly 180 185 190 Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro Ala Cys Gly Glu 195 200 205 Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro Glu Tyr Pro Asn 210 215 220 Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile Ser Val Thr Pro 225 230 235 240 Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp Leu Tyr Arg Ser 245 250 255 Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp Gly Phe Trp Arg 260 265 270 Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser Lys Leu Pro Glu Pro 275 280 285 Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe Arg Ser Ser Ser 290 295 300 Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu Ala Ile Cys Gly 305 310 315 320 Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser Pro Asn Tyr Pro 325 330 335 Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg Ile Gln Val Ser 340 345 350 Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe Glu Ile Glu Arg 355 360 365 His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg Asp Gly His Ser 370 375 380 Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr Glu Lys Pro Asp 385 390 395 400 Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys Phe Val Ser Asp 405 410 415 Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe Phe Lys Glu Val 420 425 430 Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu Gln Arg Cys Leu 435 440 445 Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro Gly Tyr Glu Leu 450 455 460 Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly Gly Phe Leu Thr 465 470 475 480 Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro Lys Glu Tyr Pro 485 490 495 Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro Thr Gln Tyr Arg 500 505 510 Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr Glu Gly Asn Asp Val Cys 515 520 525 Lys Tyr Asp Phe Val Glu Val Arg Ser Gly Leu Thr Ala Asp Ser Lys 530 535 540 Leu His Gly Lys Phe Cys Gly Ser Glu Lys Pro Glu Val Ile Thr Ser 545 550 555 560 Gln Tyr Asn Asn Met Arg Val Glu Phe Lys Ser Asp Asn Thr Val Ser 565 570 575 Lys Lys Gly Phe Lys Ala His Phe Phe Ser Asp Lys Asp Glu Cys Ser 580 585 590 Lys Asp Asn Gly Gly Cys Gln Gln Asp Cys Val Asn Thr Phe Gly Ser 595 600 605 Tyr Glu Cys Gln Cys Arg Ser Gly Phe Val Leu His Asp Asn Lys His 610 615 620 Asp Cys Lys Glu Ala Gly Cys Asp His Lys Val Thr Ser Thr Ser Gly 625 630 635 640 Thr Ile Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro Ser Lys Lys Glu 645 650 655 Cys Thr Trp Ala Ile Ser Ser Thr Pro Gly His Arg Val Lys Leu Thr 660 665 670 Phe Met Glu Met Asp Ile Glu Ser Gln Pro Glu Cys Ala Tyr Asp His 675 680 685 Leu Glu Val Phe Asp Gly Arg Asp Ala Lys Ala Pro Val Leu Gly Arg 690 695 700 Phe Cys Gly Ser Lys Lys Pro Glu Pro Val Leu Ala Thr Gly Ser Arg 705 710 715 720 Met Phe Leu Arg Phe Tyr Ser Asp Asn Ser Val Gln Arg Lys Gly Phe 725 730 735 Gln Ala Ser His Ala Thr Glu Cys Gly Gly Gln Val Arg Ala Asp Val 740 745 750 Lys Thr Lys Asp Leu Tyr Ser His Ala Gln Phe Gly Asp Asn Asn Tyr 755 760 765 Pro Gly Gly Val Asp Cys Glu Trp Val Ile Val Ala Glu Glu Gly Tyr 770 775 780 Gly Val Glu Leu Val Phe Gln Thr Phe Glu Val Glu Glu Glu Thr Asp 785 790 795 800 Cys Gly Tyr Asp Tyr Met Glu Leu Phe Asp Gly Tyr Asp Ser Thr Ala 805 810 815 Pro Arg Leu Gly Arg Tyr Cys Gly Ser Gly Pro Pro Glu Glu Val Tyr 820 825 830 Ser Ala Gly Asp Ser Val Leu Val Lys Phe His Ser Asp Asp Thr Ile 835 840 845 Thr Lys Lys Gly Phe His Leu Arg Tyr Thr Ser Thr Lys Phe Gln Asp 850 855 860 Thr Leu His Ser Arg Lys 865 870 <210> 20 <211> 2912 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the human Procollagen I N-proteinase and flanking regions <400> 20 gcgccatggc tcaattgagg agaagggcta ggagacacgc agctgatgat gattacaaca 60 ttgaagtttt gcttggtgtt gatgatagtg tggtgcaatt ccacggaaaa gagcatgttc 120 agaaatatct tttgacactt atgaatattg tgaacgaaat ctaccatgat gagtctttgg 180 gagcacacat taacgtggtt cttgtgagga ttattcttct ttcatacggt aaatctatgt 240 cacttattga gattggaaac ccttctcagt ctcttgagaa tgtgtgcaga tgggcatacc 300 ttcaacagaa gcctgatact ggacacgatg agtatcacga tcacgctatt ttccttacaa 360 ggcaggattt cggtccaagt ggaatgcaag gatatgctcc tgttactggt atgtgccacc 420 ctgttaggtc ttgtacactt aaccacgagg atggtttttc atctgctttc gtggtggctc 480 atgagacagg tcatgttttg ggaatggaac atgatggaca gggtaataga tgtggagatg 540 aagtgagact tggttcaatt atggctcctc ttgttcaagc tgcttttcat aggttccact 600 ggagtaggtg ttcacagcaa gagttgagta gataccttca ttcttacgat tgcttgcttg 660 atgatccatt tgctcatgat tggccagctt tgcctcaact tcctggattg cactactcta 720 tgaacgagca gtgcagattt gatttcggtc ttggttacat gatgtgcaca gctttcagga 780 ctttcgatcc atgcaaacag ttgtggtgtt cacacccaga taacccatat ttctgtaaaa 840 caaaaaaagg tccaccactt gatggtacta tgtgcgcacc tggaaagcac tgcttcaagg 900 gacactgcat ttggcttact cctgatattc ttaaaaggga tggatcatgg ggagcttggt 960 ctccattcgg aagttgctca agaacttgcg gaacaggtgt taagtttaga actaggcagt 1020 gcgataatcc acaccctgct aatggtggta gaacttgctc tggacttgct tacgattttc 1080 agttgtgttc taggcaagat tgccctgata gtcttgctga ttttagagaa gagcaatgta 1140 gacagtggga tctttacttt gagcacggcg acgctcagca ccactggctt ccacacgagc 1200 atagagatgc aaaagaaagg tgtcaccttt attgcgagag tagagagact ggagaggtgg 1260 tgtcaatgaa gagaatggtg cacgatggta caaggtgttc ttataaggat gcattctctt 1320 tgtgtgtgag gggagattgc aggaaagtgg gttgtgatgg agtgattgga tctagtaagc 1380 aagaagataa gtgcggagtg tgcggaggag ataactctca ttgcaaggtt gtgaaaggaa 1440 cttttacaag atcaccaaaa aaacacggtt acattaagat gttcgaaatt cctgctggag 1500 caaggcattt gcttattcag gaagtggatg caacatctca ccacttggca gtgaaaaacc 1560 ttgagactgg aaaattcatt ttgaacgagg agaacgatgt tgatgcatct agtaagactt 1620 tcattgcaat gggtgttgaa tgggagtata gggatgagga tggaagggaa acacttcaaa 1680 caatgggtcc tcttcatgga acaattactg tgttggtgat tccagtggga gatacaaggg 1740 tgtcattgac atacaagtat atgattcacg aggatagtct taacgttgat gataacaacg 1800 ttttggaaga agattctgtg gtttacgagt gggctcttaa gaaatggtca ccttgctcta 1860 agccatgtgg tggaggaagt cagttcacta agtatggttg taggaggagg cttgatcata 1920 agatggttca taggggattt tgcgcagcac ttagtaagcc aaaggcaatt aggagggctt 1980 gtaaccctca agaatgctca caaccagttt gggtgacagg agagtgggag ccatgttcac 2040 aaacatgcgg aagaactgga atgcaagtta gatcagttag atgcattcaa cctcttcatg 2100 ataacactac aagaagtgtg cacgcaaaac actgtaacga tgctaggcca gagagtagaa 2160 gagcttgctc tagggaactt tgccctggta gatggagggc aggaccttgg agtcagtgct 2220 ctgtgacatg tggaaacggt actcaggaaa gacctgttcc atgtagaact gctgatgata 2280 gtttcggaat ttgtcaggag gaaaggccag aaacagctag gacttgtaga cttggacctt 2340 gtcctaggaa tatttctgat cctagtaaaa aatcatacgt ggtgcaatgg ttgagtaggc 2400 cagatccaga ttcaccaatt aggaagattt cttcaaaagg acactgccag ggtgataaga 2460 gtattttctg cagaatggaa gttcttagta ggtactgttc tattccaggt tataacaaac 2520 tttcttgtaa gagttgcaac ttgtataaca atcttactaa cgtggagggt agaattgaac 2580 ctccaccagg aaagcacaac gatattgatg tgtttatgcc tactcttcct gtgccaacag 2640 ttgcaatgga agttagacct tctccatcta ctccacttga ggtgccactt aatgcatcaa 2700 gtactaacgc tactgaggat cacccagaga ctaacgcagt tgatgagcct tataagattc 2760 acggacttga ggatgaggtt cagccaccaa accttattcc taggaggcca agtccttacg 2820 aaaaaactag aaatcagagg attcaggagc ttattgatga gatgaggaaa aaggagatgc 2880 ttggaaagtt ctaatgagct cgcggccgca tc 2912 <210> 21 <211> 962 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence containing the human Procollagen I N-proteinase and flanking regions <400> 21 Met Ala Gln Leu Arg Arg Arg Ala Arg Arg His Ala Ala Asp Asp Asp 1 5 10 15 Tyr Asn Ile Glu Val Leu Leu Gly Val Asp Asp Ser Val Val Gln Phe 20 25 30 His Gly Lys Glu His Val Gln Lys Tyr Leu Leu Thr Leu Met Asn Ile 35 40 45 Val Asn Glu Ile Tyr His Asp Glu Ser Leu Gly Ala His Ile Asn Val 50 55 60 Val Leu Val Arg Ile Ile Leu Leu Ser Tyr Gly Lys Ser Met Ser Leu 65 70 75 80 Ile Glu Ile Gly Asn Pro Ser Gln Ser Leu Glu Asn Val Cys Arg Trp 85 90 95 Ala Tyr Leu Gln Gln Lys Pro Asp Thr Gly His Asp Glu Tyr His Asp 100 105 110 His Ala Ile Phe Leu Thr Arg Gln Asp Phe Gly Pro Ser Gly Met Gln 115 120 125 Gly Tyr Ala Pro Val Thr Gly Met Cys His Pro Val Arg Ser Cys Thr 130 135 140 Leu Asn His Glu Asp Gly Phe Ser Ser Ala Phe Val Val Ala His Glu 145 150 155 160 Thr Gly His Val Leu Gly Met Glu His Asp Gly Gln Gly Asn Arg Cys 165 170 175 Gly Asp Glu Val Arg Leu Gly Ser Ile Met Ala Pro Leu Val Gln Ala 180 185 190 Ala Phe His Arg Phe His Trp Ser Arg Cys Ser Gln Gln Glu Leu Ser 195 200 205 Arg Tyr Leu His Ser Tyr Asp Cys Leu Leu Asp Asp Pro Phe Ala His 210 215 220 Asp Trp Pro Ala Leu Pro Gln Leu Pro Gly Leu His Tyr Ser Met Asn 225 230 235 240 Glu Gln Cys Arg Phe Asp Phe Gly Leu Gly Tyr Met Met Cys Thr Ala 245 250 255 Phe Arg Thr Phe Asp Pro Cys Lys Gln Leu Trp Cys Ser His Pro Asp 260 265 270 Asn Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro Pro Leu Asp Gly Thr 275 280 285 Met Cys Ala Pro Gly Lys His Cys Phe Lys Gly His Cys Ile Trp Leu 290 295 300 Thr Pro Asp Ile Leu Lys Arg Asp Gly Ser Trp Gly Ala Trp Ser Pro 305 310 315 320 Phe Gly Ser Cys Ser Arg Thr Cys Gly Thr Gly Val Lys Phe Arg Thr 325 330 335 Arg Gln Cys Asp Asn Pro His Pro Ala Asn Gly Gly Arg Thr Cys Ser 340 345 350 Gly Leu Ala Tyr Asp Phe Gln Leu Cys Ser Arg Gln Asp Cys Pro Asp 355 360 365 Ser Leu Ala Asp Phe Arg Glu Glu Gln Cys Arg Gln Trp Asp Leu Tyr 370 375 380 Phe Glu His Gly Asp Ala Gln His His Trp Leu Pro His Glu His Arg 385 390 395 400 Asp Ala Lys Glu Arg Cys His Leu Tyr Cys Glu Ser Arg Glu Thr Gly 405 410 415 Glu Val Val Ser Met Lys Arg Met Val His Asp Gly Thr Arg Cys Ser 420 425 430 Tyr Lys Asp Ala Phe Ser Leu Cys Val Arg Gly Asp Cys Arg Lys Val 435 440 445 Gly Cys Asp Gly Val Ile Gly Ser Ser Lys Gln Glu Asp Lys Cys Gly 450 455 460 Val Cys Gly Gly Asp Asn Ser His Cys Lys Val Val Lys Gly Thr Phe 465 470 475 480 Thr Arg Ser Pro Lys Lys His Gly Tyr Ile Lys Met Phe Glu Ile Pro 485 490 495 Ala Gly Ala Arg His Leu Leu Ile Gln Glu Val Asp Ala Thr Ser His 500 505 510 His Leu Ala Val Lys Asn Leu Glu Thr Gly Lys Phe Ile Leu Asn Glu 515 520 525 Glu Asn Asp Val Asp Ala Ser Ser Lys Thr Phe Ile Ala Met Gly Val 530 535 540 Glu Trp Glu Tyr Arg Asp Glu Asp Gly Arg Glu Thr Leu Gln Thr Met 545 550 555 560 Gly Pro Leu His Gly Thr Ile Thr Val Leu Val Ile Pro Val Gly Asp 565 570 575 Thr Arg Val Ser Leu Thr Tyr Lys Tyr Met Ile His Glu Asp Ser Leu 580 585 590 Asn Val Asp Asp Asn Asn Val Leu Glu Glu Asp Ser Val Val Tyr Glu 595 600 605 Trp Ala Leu Lys Lys Trp Ser Pro Cys Ser Lys Pro Cys Gly Gly Gly 610 615 620 Ser Gln Phe Thr Lys Tyr Gly Cys Arg Arg Arg Leu Asp His Lys Met 625 630 635 640 Val His Arg Gly Phe Cys Ala Ala Leu Ser Lys Pro Lys Ala Ile Arg 645 650 655 Arg Ala Cys Asn Pro Gln Glu Cys Ser Gln Pro Val Trp Val Thr Gly 660 665 670 Glu Trp Glu Pro Cys Ser Gln Thr Cys Gly Arg Thr Gly Met Gln Val 675 680 685 Arg Ser Val Arg Cys Ile Gln Pro Leu His Asp Asn Thr Thr Arg Ser 690 695 700 Val His Ala Lys His Cys Asn Asp Ala Arg Pro Glu Ser Arg Arg Ala 705 710 715 720 Cys Ser Arg Glu Leu Cys Pro Gly Arg Trp Arg Ala Gly Pro Trp Ser 725 730 735 Gln Cys Ser Val Thr Cys Gly Asn Gly Thr Gln Glu Arg Pro Val Pro 740 745 750 Cys Arg Thr Ala Asp Asp Ser Phe Gly Ile Cys Gln Glu Glu Arg Pro 755 760 765 Glu Thr Ala Arg Thr Cys Arg Leu Gly Pro Cys Pro Arg Asn Ile Ser 770 775 780 Asp Pro Ser Lys Lys Ser Tyr Val Val Gln Trp Leu Ser Arg Pro Asp 785 790 795 800 Pro Asp Ser Pro Ile Arg Lys Ile Ser Ser Lys Gly His Cys Gln Gly 805 810 815 Asp Lys Ser Ile Phe Cys Arg Met Glu Val Leu Ser Arg Tyr Cys Ser 820 825 830 Ile Pro Gly Tyr Asn Lys Leu Ser Cys Lys Ser Cys Asn Leu Tyr Asn 835 840 845 Asn Leu Thr Asn Val Glu Gly Arg Ile Glu Pro Pro Pro Gly Lys His 850 855 860 Asn Asp Ile Asp Val Phe Met Pro Thr Leu Pro Val Pro Thr Val Ala 865 870 875 880 Met Glu Val Arg Pro Ser Pro Ser Thr Pro Leu Glu Val Pro Leu Asn 885 890 895 Ala Ser Ser Thr Asn Ala Thr Glu Asp His Pro Glu Thr Asn Ala Val 900 905 910 Asp Glu Pro Tyr Lys Ile His Gly Leu Glu Asp Glu Val Gln Pro Pro 915 920 925 Asn Leu Ile Pro Arg Arg Pro Ser Pro Tyr Glu Lys Thr Arg Asn Gln 930 935 940 Arg Ile Gln Glu Leu Ile Asp Glu Met Arg Lys Lys Glu Met Leu Gly 945 950 955 960 Lys Phe <210> 22 <211> 2888 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Lysyl hydroxylase 3 and flanking regions <400> 22 gcgaattcgc tagctatcac tgaaaagaca gcaagacaat ggtgtctcga tgcaccagaa 60 ccacatcttt gcagcagatg tgaagcagcc agagtggtcc acaagacgca ctcagaaaag 120 gcatcttcta ccgacacaga aaaagacaac cacagctcat catccaacat gtagactgtc 180 gttatgcgtc ggctgaagat aagactgacc ccaggccagc actaaagaag aaataatgca 240 agtggtccta gctccacttt agctttaata attatgtttc attattattc tctgcttttg 300 ctctctatat aaagagcttg tattttcatt tgaaggcaga ggcgaacaca cacacagaac 360 ctccctgctt acaaaccaga tcttaaacca tggctcacgc tagggttttg cttcttgctc 420 ttgctgttct tgctactgct gctgttgctg tggcttcttc aagttctttc gctgattcta 480 acccaattag gccagtgact gatagagctg cttctactct tgctcaattg agatctatgt 540 ctgatagacc aaggggaagg gatccagtta atccagagaa gttgcttgtg attactgtgg 600 ctactgctga gactgaagga taccttagat tccttaggag tgctgagttc ttcaactaca 660 ctgtgaggac tcttggactt ggagaagaat ggaggggagg agatgttgct agaactgttg 720 gaggaggaca gaaagtgaga tggcttaaga aagagatgga gaagtacgct gatagggagg 780 atatgattat tatgttcgtg gattcttacg atgtgattct tgctggatct ccaactgagc 840 ttttgaagaa attcgttcag tctggatcta ggcttctttt ctctgctgag tctttttgtt 900 ggccagaatg gggacttgct gagcaatatc cagaagtggg aactggaaag agattcctta 960 actctggagg attcattgga ttcgctacta ctattcacca gattgtgagg cagtggaagt 1020 acaaggatga cgatgatgat cagcttttct acactaggct ttaccttgat ccaggactta 1080 gggagaagtt gtctcttaac cttgatcaca agtctaggat tttccagaac cttaacggtg 1140 ctcttgatga ggttgtgctt aagttcgata ggaacagagt gaggattagg aacgtggctt 1200 acgatactct tcctattgtg gtgcatggaa acggaccaac aaaactccag cttaactacc 1260 ttggaaacta cgttccaaac ggatggactc cagaaggagg atgtggattc tgcaatcagg 1320 ataggagaac tcttccagga ggacaaccac caccaagagt tttccttgct gtgttcgttg 1380 aacagccaac tccattcctt ccaagattcc ttcagaggct tcttcttttg gattacccac 1440 cagatagggt gacacttttc cttcacaaca acgaggtttt ccacgagcca cacattgctg 1500 attcttggcc acagcttcag gatcatttct ctgctgtgaa gttggttggt ccagaagaag 1560 ctctttctcc aggagaagct agggatatgg ctatggattt gtgcaggcag gatccagagt 1620 gcgagttcta cttctctctt gatgctgatg ctgtgcttac taaccttcag actcttagga 1680 ttcttattga ggagaacagg aaagtgattg ctccaatgct ttctaggcac ggaaagttgt 1740 ggtctaattt ctggggtgct ctttctcctg atgagtacta cgctagatca gaggactacg 1800 tggagcttgt tcagagaaag agagtgggag tttggaacgt tccttatatt tctcaggctt 1860 acgtgattag gggagatact cttaggatgg agcttccaca gagggatgtt ttctctggat 1920 ctgatactga tccagatatg gctttctgca agtctttcag ggataaggga attttccttc 1980 acctttctaa ccagcatgag ttcggaagat tgcttgctac ttcaagatac gatactgagc 2040 accttcatcc tgatctttgg cagattttcg ataacccagt ggattggaag gagcagtaca 2100 ttcacgagaa ctactctagg gctcttgaag gagaaggaat tgtggagcaa ccatgcccag 2160 atgtttactg gttcccactt ctttctgagc aaatgtgcga tgagcttgtt gctgagatgg 2220 agcattacgg acaatggagt ggaggtagac atgaggattc taggcttgct ggaggatacg 2280 agaacgttcc aactgtggat attcacatga agcaagtggg atacgaggat caatggcttc 2340 agcttcttag gacttatgtg ggaccaatga ctgagtctct tttcccagga taccacacta 2400 aggctagggc tgttatgaac ttcgttgtga ggtatcgtcc agatgagcaa ccatctctta 2460 ggccacacca cgattcttct actttcactc ttaacgtggc tcttaaccac aagggacttg 2520 attatgaggg aggaggatgc cgtttcctta gatacgattg cgtgatttct tcaccaagaa 2580 agggatgggc tcttcttcat ccaggaaggc ttactcatta ccacgaggga cttccaacta 2640 cttggggaac tagatatatt atggtgtctt tcgtggatcc atgactgctt taatgagata 2700 tgcgagacgc ctatgatcgc atgatatttg ctttcaattc tgttgtgcac gttgtaaaaa 2760 acctgagcat gtgtagctca gatccttacc gccggtttcg gttcattcta atgaatatat 2820 cacccgttac tatcgtattt ttatgaataa tattctccgt tcaatttact gattgtccag 2880 aattcgcg 2888 <210> 23 <211> 764 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence containing the vacuolar signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Lysyl hydroxylase 3 and flanking regions <400> 23 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Arg 35 40 45 Ser Met Ser Asp Arg Pro Arg Gly Arg Asp Pro Val Asn Pro Glu Lys 50 55 60 Leu Leu Val Ile Thr Val Ala Thr Ala Glu Thr Glu Gly Tyr Leu Arg 65 70 75 80 Phe Leu Arg Ser Ala Glu Phe Phe Asn Tyr Thr Val Arg Thr Leu Gly 85 90 95 Leu Gly Glu Glu Trp Arg Gly Gly Asp Val Ala Arg Thr Val Gly Gly 100 105 110 Gly Gln Lys Val Arg Trp Leu Lys Lys Glu Met Glu Lys Tyr Ala Asp 115 120 125 Arg Glu Asp Met Ile Ile Met Phe Val Asp Ser Tyr Asp Val Ile Leu 130 135 140 Ala Gly Ser Pro Thr Glu Leu Leu Lys Lys Phe Val Gln Ser Gly Ser 145 150 155 160 Arg Leu Leu Phe Ser Ala Glu Ser Phe Cys Trp Pro Glu Trp Gly Leu 165 170 175 Ala Glu Gln Tyr Pro Glu Val Gly Thr Gly Lys Arg Phe Leu Asn Ser 180 185 190 Gly Gly Phe Ile Gly Phe Ala Thr Thr Ile His Gln Ile Val Arg Gln 195 200 205 Trp Lys Tyr Lys Asp Asp Asp Asp Asp Gln Leu Phe Tyr Thr Arg Leu 210 215 220 Tyr Leu Asp Pro Gly Leu Arg Glu Lys Leu Ser Leu Asn Leu Asp His 225 230 235 240 Lys Ser Arg Ile Phe Gln Asn Leu Asn Gly Ala Leu Asp Glu Val Val 245 250 255 Leu Lys Phe Asp Arg Asn Arg Val Arg Ile Arg Asn Val Ala Tyr Asp 260 265 270 Thr Leu Pro Ile Val Val His Gly Asn Gly Pro Thr Lys Leu Gln Leu 275 280 285 Asn Tyr Leu Gly Asn Tyr Val Pro Asn Gly Trp Thr Pro Glu Gly Gly 290 295 300 Cys Gly Phe Cys Asn Gln Asp Arg Arg Thr Leu Pro Gly Gly Gln Pro 305 310 315 320 Pro Pro Arg Val Phe Leu Ala Val Phe Val Glu Gln Pro Thr Pro Phe 325 330 335 Leu Pro Arg Phe Leu Gln Arg Leu Leu Leu Leu Asp Tyr Pro Pro Asp 340 345 350 Arg Val Thr Leu Phe Leu His Asn Asn Glu Val Phe His Glu Pro His 355 360 365 Ile Ala Asp Ser Trp Pro Gln Leu Gln Asp His Phe Ser Ala Val Lys 370 375 380 Leu Val Gly Pro Glu Glu Ala Leu Ser Pro Gly Glu Ala Arg Asp Met 385 390 395 400 Ala Met Asp Leu Cys Arg Gln Asp Pro Glu Cys Glu Phe Tyr Phe Ser 405 410 415 Leu Asp Ala Asp Ala Val Leu Thr Asn Leu Gln Thr Leu Arg Ile Leu 420 425 430 Ile Glu Glu Asn Arg Lys Val Ile Ala Pro Met Leu Ser Arg His Gly 435 440 445 Lys Leu Trp Ser Asn Phe Trp Gly Ala Leu Ser Pro Asp Glu Tyr Tyr 450 455 460 Ala Arg Ser Glu Asp Tyr Val Glu Leu Val Gln Arg Lys Arg Val Gly 465 470 475 480 Val Trp Asn Val Pro Tyr Ile Ser Gln Ala Tyr Val Ile Arg Gly Asp 485 490 495 Thr Leu Arg Met Glu Leu Pro Gln Arg Asp Val Phe Ser Gly Ser Asp 500 505 510 Thr Asp Pro Asp Met Ala Phe Cys Lys Ser Phe Arg Asp Lys Gly Ile 515 520 525 Phe Leu His Leu Ser Asn Gln His Glu Phe Gly Arg Leu Leu Ala Thr 530 535 540 Ser Arg Tyr Asp Thr Glu His Leu His Pro Asp Leu Trp Gln Ile Phe 545 550 555 560 Asp Asn Pro Val Asp Trp Lys Glu Gln Tyr Ile His Glu Asn Tyr Ser 565 570 575 Arg Ala Leu Glu Gly Glu Gly Ile Val Glu Gln Pro Cys Pro Asp Val 580 585 590 Tyr Trp Phe Pro Leu Leu Ser Glu Gln Met Cys Asp Glu Leu Val Ala 595 600 605 Glu Met Glu His Tyr Gly Gln Trp Ser Gly Gly Arg His Glu Asp Ser 610 615 620 Arg Leu Ala Gly Gly Tyr Glu Asn Val Pro Thr Val Asp Ile His Met 625 630 635 640 Lys Gln Val Gly Tyr Glu Asp Gln Trp Leu Gln Leu Leu Arg Thr Tyr 645 650 655 Val Gly Pro Met Thr Glu Ser Leu Phe Pro Gly Tyr His Thr Lys Ala 660 665 670 Arg Ala Val Met Asn Phe Val Val Arg Tyr Arg Pro Asp Glu Gln Pro 675 680 685 Ser Leu Arg Pro His His Asp Ser Ser Thr Phe Thr Leu Asn Val Ala 690 695 700 Leu Asn His Lys Gly Leu Asp Tyr Glu Gly Gly Gly Cys Arg Phe Leu 705 710 715 720 Arg Tyr Asp Cys Val Ile Ser Ser Pro Arg Lys Gly Trp Ala Leu Leu 725 730 735 His Pro Gly Arg Leu Thr His Tyr His Glu Gly Leu Pro Thr Thr Trp 740 745 750 Gly Thr Arg Tyr Ile Met Val Ser Phe Val Asp Pro 755 760 <210> 24 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Vacuole signal sequence of barley gene for Thiol protease aleurain precursor <400> 24 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala 35 40 45 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA oligonucleotide <400> 25 atcaccagga gaacagggac catc 24 <210> 26 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA oligonucleotide <400> 26 tccacttcca aatctctatc cctaacaac 29 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA oligonucleotide <400> 27 aggcattaga ggcgataagg gag 23 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Single strand DNA oligonucleotide <400> 28 tcaatccaat aatagccact tgaccac 27 <210> 29 <211> 102 <212> DNA <213> Artificial Sequence <220> <223> pBINPLUS multiple cloning site <400> 29 atgaccatga ttacgccaag ctggcgcgcc aagcttgcat gcctgcaggt cgactctaga 60 ggatccccgg gtaccgagct cgaattctta attaacaatt ca 102 <210> 30 <211> 1464 <212> PRT <213> Homo sapiens <400> 30 Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr 1 5 10 15 Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp 20 25 30 Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His 35 40 45 Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp 50 55 60 Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn 65 70 75 80 Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro 85 90 95 Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly 100 105 110 Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro 115 120 125 Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro 130 135 140 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala 145 150 155 160 Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser 165 170 175 Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro 180 185 190 Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro 195 200 205 Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly 210 215 220 Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg 225 230 235 240 Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro 245 250 255 Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly 260 265 270 Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu 275 280 285 Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg 290 295 300 Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly 305 310 315 320 Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro 325 330 335 Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys 340 345 350 Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly 355 360 365 Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro 370 375 380 Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn 385 390 395 400 Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly 405 410 415 Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn 420 425 430 Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys 435 440 445 Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly 450 455 460 Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu 465 470 475 480 Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro 485 490 495 Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly 500 505 510 Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg 515 520 525 Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro 530 535 540 Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly 545 550 555 560 Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln 565 570 575 Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro 580 585 590 Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly 595 600 605 Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro 610 615 620 Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro 625 630 635 640 Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly 645 650 655 Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro 660 665 670 Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln 675 680 685 Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly 690 695 700 Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser 705 710 715 720 Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala 725 730 735 Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly 740 745 750 Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro 755 760 765 Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser 770 775 780 Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly 785 790 795 800 Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro 805 810 815 Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala 820 825 830 Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly 835 840 845 Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala 850 855 860 Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala 865 870 875 880 Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly 885 890 895 Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu 900 905 910 Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro 915 920 925 Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly 930 935 940 Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val 945 950 955 960 Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro 965 970 975 Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly 980 985 990 Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro 995 1000 1005 Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Gly Ala Glu Gly Ser 1010 1015 1020 Pro Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu 1025 1030 1035 Thr Gly Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala 1040 1045 1050 Pro Gly Pro Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu 1055 1060 1065 Thr Gly Pro Ala Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala 1070 1075 1080 Arg Gly Pro Ala Gly Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu 1085 1090 1095 Thr Gly Glu Gln Gly Asp Arg Gly Ile Lys Gly His Arg Gly Phe 1100 1105 1110 Ser Gly Leu Gln Gly Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu 1115 1120 1125 Gln Gly Pro Ser Gly Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro 1130 1135 1140 Pro Gly Ser Ala Gly Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu 1145 1150 1155 Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp 1160 1165 1170 Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro 1175 1180 1185 Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln 1190 1195 1200 Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala 1205 1210 1215 Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp Thr 1220 1225 1230 Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro 1235 1240 1245 Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys 1250 1255 1260 Met Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro 1265 1270 1275 Asn Gln Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met 1280 1285 1290 Glu Thr Gly Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala 1295 1300 1305 Gln Lys Asn Trp Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His 1310 1315 1320 Val Trp Phe Gly Glu Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr 1325 1330 1335 Gly Gly Gln Gly Ser Asp Pro Ala Asp Val Ala Ile Gln Leu Thr 1340 1345 1350 Phe Leu Arg Leu Met Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr 1355 1360 1365 His Cys Lys Asn Ser Val Ala Tyr Met Asp Gln Gln Thr Gly Asn 1370 1375 1380 Leu Lys Lys Ala Leu Leu Leu Gln Gly Ser Asn Glu Ile Glu Ile 1385 1390 1395 Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr Ser Val Thr Val Asp 1400 1405 1410 Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys Thr Val Ile Glu 1415 1420 1425 Tyr Lys Thr Thr Lys Thr Ser Arg Leu Pro Ile Ile Asp Val Ala 1430 1435 1440 Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe Asp Val 1445 1450 1455 Gly Pro Val Cys Phe Leu 1460 <210> 31 <211> 1366 <212> PRT <213> Homo sapiens <400> 31 Met Leu Ser Phe Val Asp Thr Arg Thr Leu Leu Leu Leu Ala Val Thr 1 5 10 15 Leu Cys Leu Ala Thr Cys Gln Ser Leu Gln Glu Glu Thr Val Arg Lys 20 25 30 Gly Pro Ala Gly Asp Arg Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly 35 40 45 Pro Pro Gly Arg Asp Gly Glu Asp Gly Pro Thr Gly Pro Pro Gly Pro 50 55 60 Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala Ala Gln 65 70 75 80 Tyr Asp Gly Lys Gly Val Gly Leu Gly Pro Gly Pro Met Gly Leu Met 85 90 95 Gly Pro Arg Gly Pro Pro Gly Ala Ala Gly Ala Pro Gly Pro Gln Gly 100 105 110 Phe Gln Gly Pro Ala Gly Glu Pro Gly Glu Pro Gly Gln Thr Gly Pro 115 120 125 Ala Gly Ala Arg Gly Pro Ala Gly Pro Pro Gly Lys Ala Gly Glu Asp 130 135 140 Gly His Pro Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Val Val Gly 145 150 155 160 Pro Gln Gly Ala Arg Gly Phe Pro Gly Thr Pro Gly Leu Pro Gly Phe 165 170 175 Lys Gly Ile Arg Gly His Asn Gly Leu Asp Gly Leu Lys Gly Gln Pro 180 185 190 Gly Ala Pro Gly Val Lys Gly Glu Pro Gly Ala Pro Gly Glu Asn Gly 195 200 205 Thr Pro Gly Gln Thr Gly Ala Arg Gly Leu Pro Gly Glu Arg Gly Arg 210 215 220 Val Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Ser Asp Gly Ser Val 225 230 235 240 Gly Pro Val Gly Pro Ala Gly Pro Ile Gly Ser Ala Gly Pro Pro Gly 245 250 255 Phe Pro Gly Ala Pro Gly Pro Lys Gly Glu Ile Gly Ala Val Gly Asn 260 265 270 Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Glu Val Gly Leu Pro 275 280 285 Gly Leu Ser Gly Pro Val Gly Pro Pro Gly Asn Pro Gly Ala Asn Gly 290 295 300 Leu Thr Gly Ala Lys Gly Ala Ala Gly Leu Pro Gly Val Ala Gly Ala 305 310 315 320 Pro Gly Leu Pro Gly Pro Arg Gly Ile Pro Gly Pro Val Gly Ala Ala 325 330 335 Gly Ala Thr Gly Ala Arg Gly Leu Val Gly Glu Pro Gly Pro Ala Gly 340 345 350 Ser Lys Gly Glu Ser Gly Asn Lys Gly Glu Pro Gly Ser Ala Gly Pro 355 360 365 Gln Gly Pro Pro Gly Pro Ser Gly Glu Glu Gly Lys Arg Gly Pro Asn 370 375 380 Gly Glu Ala Gly Ser Ala Gly Pro Pro Gly Pro Pro Gly Leu Arg Gly 385 390 395 400 Ser Pro Gly Ser Arg Gly Leu Pro Gly Ala Asp Gly Arg Ala Gly Val 405 410 415 Met Gly Pro Pro Gly Ser Arg Gly Ala Ser Gly Pro Ala Gly Val Arg 420 425 430 Gly Pro Asn Gly Asp Ala Gly Arg Pro Gly Glu Pro Gly Leu Met Gly 435 440 445 Pro Arg Gly Leu Pro Gly Ser Pro Gly Asn Ile Gly Pro Ala Gly Lys 450 455 460 Glu Gly Pro Val Gly Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile 465 470 475 480 Gly Pro Ala Gly Ala Arg Gly Glu Pro Gly Asn Ile Gly Phe Pro Gly 485 490 495 Pro Lys Gly Pro Thr Gly Asp Pro Gly Lys Asn Gly Asp Lys Gly His 500 505 510 Ala Gly Leu Ala Gly Ala Arg Gly Ala Pro Gly Pro Asp Gly Asn Asn 515 520 525 Gly Ala Gln Gly Pro Pro Gly Pro Gln Gly Val Gln Gly Gly Lys Gly 530 535 540 Glu Gln Gly Pro Ala Gly Pro Pro Gly Phe Gln Gly Leu Pro Gly Pro 545 550 555 560 Ser Gly Pro Ala Gly Glu Val Gly Lys Pro Gly Glu Arg Gly Leu His 565 570 575 Gly Glu Phe Gly Leu Pro Gly Pro Ala Gly Pro Arg Gly Glu Arg Gly 580 585 590 Pro Pro Gly Glu Ser Gly Ala Ala Gly Pro Thr Gly Pro Ile Gly Ser 595 600 605 Arg Gly Pro Ser Gly Pro Pro Gly Pro Asp Gly Asn Lys Gly Glu Pro 610 615 620 Gly Val Val Gly Ala Val Gly Thr Ala Gly Pro Ser Gly Pro Ser Gly 625 630 635 640 Leu Pro Gly Glu Arg Gly Ala Ala Gly Ile Pro Gly Gly Lys Gly Glu 645 650 655 Lys Gly Glu Pro Gly Leu Arg Gly Glu Ile Gly Asn Pro Gly Arg Asp 660 665 670 Gly Ala Arg Gly Ala Pro Gly Ala Val Gly Ala Pro Gly Pro Ala Gly 675 680 685 Ala Thr Gly Asp Arg Gly Glu Ala Gly Ala Ala Gly Pro Ala Gly Pro 690 695 700 Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg Gly Glu Val Gly Pro Ala 705 710 715 720 Gly Pro Asn Gly Phe Ala Gly Pro Ala Gly Ala Ala Gly Gln Pro Gly 725 730 735 Ala Lys Gly Glu Arg Gly Ala Lys Gly Pro Lys Gly Glu Asn Gly Val 740 745 750 Val Gly Pro Thr Gly Pro Val Gly Ala Ala Gly Pro Ala Gly Pro Asn 755 760 765 Gly Pro Pro Gly Pro Ala Gly Ser Arg Gly Asp Gly Gly Pro Pro Gly 770 775 780 Met Thr Gly Phe Pro Gly Ala Ala Gly Arg Thr Gly Pro Pro Gly Pro 785 790 795 800 Ser Gly Ile Ser Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys Glu 805 810 815 Gly Leu Arg Gly Pro Arg Gly Asp Gln Gly Pro Val Gly Arg Thr Gly 820 825 830 Glu Val Gly Ala Val Gly Pro Pro Gly Phe Ala Gly Glu Lys Gly Pro 835 840 845 Ser Gly Glu Ala Gly Thr Ala Gly Pro Pro Gly Thr Pro Gly Pro Gln 850 855 860 Gly Leu Leu Gly Ala Pro Gly Ile Leu Gly Leu Pro Gly Ser Arg Gly 865 870 875 880 Glu Arg Gly Leu Pro Gly Val Ala Gly Ala Val Gly Glu Pro Gly Pro 885 890 895 Leu Gly Ile Ala Gly Pro Pro Gly Ala Arg Gly Pro Pro Gly Ala Val 900 905 910 Gly Ser Pro Gly Val Asn Gly Ala Pro Gly Glu Ala Gly Arg Asp Gly 915 920 925 Asn Pro Gly Asn Asp Gly Pro Pro Gly Arg Asp Gly Gln Pro Gly His 930 935 940 Lys Gly Glu Arg Gly Tyr Pro Gly Asn Ile Gly Pro Val Gly Ala Ala 945 950 955 960 Gly Ala Pro Gly Pro His Gly Pro Val Gly Pro Ala Gly Lys His Gly 965 970 975 Asn Arg Gly Glu Thr Gly Pro Ser Gly Pro Val Gly Pro Ala Gly Ala 980 985 990 Val Gly Pro Arg Gly Pro Ser Gly Pro Gln Gly Ile Arg Gly Asp Lys 995 1000 1005 Gly Glu Pro Gly Glu Lys Gly Pro Arg Gly Leu Pro Gly Leu Lys 1010 1015 1020 Gly His Asn Gly Leu Gln Gly Leu Pro Gly Ile Ala Gly His His 1025 1030 1035 Gly Asp Gln Gly Ala Pro Gly Ser Val Gly Pro Ala Gly Pro Arg 1040 1045 1050 Gly Pro Ala Gly Pro Ser Gly Pro Ala Gly Lys Asp Gly Arg Thr 1055 1060 1065 Gly His Pro Gly Thr Val Gly Pro Ala Gly Ile Arg Gly Pro Gln 1070 1075 1080 Gly His Gln Gly Pro Ala Gly Pro Pro Gly Pro Pro Gly Pro Pro 1085 1090 1095 Gly Pro Pro Gly Val Ser Gly Gly Gly Tyr Asp Phe Gly Tyr Asp 1100 1105 1110 Gly Asp Phe Tyr Arg Ala Asp Gln Pro Arg Ser Ala Pro Ser Leu 1115 1120 1125 Arg Pro Lys Asp Tyr Glu Val Asp Ala Thr Leu Lys Ser Leu Asn 1130 1135 1140 Asn Gln Ile Glu Thr Leu Leu Thr Pro Glu Gly Ser Arg Lys Asn 1145 1150 1155 Pro Ala Arg Thr Cys Arg Asp Leu Arg Leu Ser His Pro Glu Trp 1160 1165 1170 Ser Ser Gly Tyr Tyr Trp Ile Asp Pro Asn Gln Gly Cys Thr Met 1175 1180 1185 Asp Ala Ile Lys Val Tyr Cys Asp Phe Ser Thr Gly Glu Thr Cys 1190 1195 1200 Ile Arg Ala Gln Pro Glu Asn Ile Pro Ala Lys Asn Trp Tyr Arg 1205 1210 1215 Ser Ser Lys Asp Lys Lys His Val Trp Leu Gly Glu Thr Ile Asn 1220 1225 1230 Ala Gly Ser Gln Phe Glu Tyr Asn Val Glu Gly Val Thr Ser Lys 1235 1240 1245 Glu Met Ala Thr Gln Leu Ala Phe Met Arg Leu Leu Ala Asn Tyr 1250 1255 1260 Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Ile Ala Tyr 1265 1270 1275 Met Asp Glu Glu Thr Gly Asn Leu Lys Lys Ala Val Ile Leu Gln 1280 1285 1290 Gly Ser Asn Asp Val Glu Leu Val Ala Glu Gly Asn Ser Arg Phe 1295 1300 1305 Thr Tyr Thr Val Leu Val Asp Gly Cys Ser Lys Lys Thr Asn Glu 1310 1315 1320 Trp Gly Lys Thr Ile Ile Glu Tyr Lys Thr Asn Lys Pro Ser Arg 1325 1330 1335 Leu Pro Phe Leu Asp Ile Ala Pro Leu Asp Ile Gly Gly Ala Asp 1340 1345 1350 Gln Glu Phe Phe Val Asp Ile Gly Pro Val Cys Phe Lys 1355 1360 1365 <210> 32 <211> 5927 <212> DNA <213> Homo sapiens <400> 32 tcgtcggagc agacgggagt ttctcctcgg ggtcggagca ggaggcacgc ggagtgtgag 60 gccacgcatg agcggacgct aaccccctcc ccagccacaa agagtctaca tgtctagggt 120 ctagacatgt tcagctttgt ggacctccgg ctcctgctcc tcttagcggc caccgccctc 180 ctgacgcacg gccaagagga aggccaagtc gagggccaag acgaagacat cccaccaatc 240 acctgcgtac agaacggcct caggtaccat gaccgagacg tgtggaaacc cgagccctgc 300 cggatctgcg tctgcgacaa cggcaaggtg ttgtgcgatg acgtgatctg tgacgagacc 360 aagaactgcc ccggcgccga agtccccgag ggcgagtgct gtcccgtctg ccccgacggc 420 tcagagtcac ccaccgacca agaaaccacc ggcgtcgagg gacccaaggg agacactggc 480 ccccgaggcc caaggggacc cgcaggcccc cctggccgag atggcatccc tggacagcct 540 ggacttcccg gaccccccgg accccccgga cctcccggac cccctggcct cggaggaaac 600 tttgctcccc agctgtctta tggctatgat gagaaatcaa ccggaggaat ttccgtgcct 660 ggccccatgg gtccctctgg tcctcgtggt ctccctggcc cccctggtgc acctggtccc 720 caaggcttcc aaggtccccc tggtgagcct ggcgagcctg gagcttcagg tcccatgggt 780 ccccgaggtc ccccaggtcc ccctggaaag aatggagatg atggggaagc tggaaaacct 840 ggtcgtcctg gtgagcgtgg gcctcctggg cctcagggtg ctcgaggatt gcccggaaca 900 gctggcctcc ctggaatgaa gggacacaga ggtttcagtg gtttggatgg tgccaaggga 960 gatgctggtc ctgctggtcc taagggtgag cctggcagcc ctggtgaaaa tggagctcct 1020 ggtcagatgg gcccccgtgg cctgcctggt gagagaggtc gccctggagc ccctggccct 1080 gctggtgctc gtggaaatga tggtgctact ggtgctgccg ggccccctgg tcccaccggc 1140 cccgctggtc ctcctggctt ccctggtgct gttggtgcta agggtgaagc tggtccccaa 1200 gggccccgag gctctgaagg tccccagggt gtgcgtggtg agcctggccc ccctggccct 1260 gctggtgctg ctggccctgc tggaaaccct ggtgctgatg gacagcctgg tgctaaaggt 1320 gccaatggtg ctcctggtat tgctggtgct cctggcttcc ctggtgcccg aggcccctct 1380 ggaccccagg gccccggcgg ccctcctggt cccaagggta acagcggtga acctggtgct 1440 cctggcagca aaggagacac tggtgctaag ggagagcctg gccctgttgg tgttcaagga 1500 ccccctggcc ctgctggaga ggaaggaaag cgaggagctc gaggtgaacc cggacccact 1560 ggcctgcccg gaccccctgg cgagcgtggt ggacctggta gccgtggttt ccctggcgca 1620 gatggtgttg ctggtcccaa gggtcccgct ggtgaacgtg gttctcctgg ccctgctggc 1680 cccaaaggat ctcctggtga agctggtcgt cccggtgaag ctggtctgcc tggtgccaag 1740 ggtctgactg gaagccctgg cagccctggt cctgatggca aaactggccc ccctggtccc 1800 gccggtcaag atggtcgccc cggaccccca ggcccacctg gtgcccgtgg tcaggctggt 1860 gtgatgggat tccctggacc taaaggtgct gctggagagc ccggcaaggc tggagagcga 1920 ggtgttcccg gaccccctgg cgctgtcggt cctgctggca aagatggaga ggctggagct 1980 cagggacccc ctggccctgc tggtcccgct ggcgagagag gtgaacaagg ccctgctggc 2040 tcccccggat tccagggtct ccctggtcct gctggtcctc caggtgaagc aggcaaacct 2100 ggtgaacagg gtgttcctgg agaccttggc gcccctggcc cctctggagc aagaggcgag 2160 agaggtttcc ctggcgagcg tggtgtgcaa ggtccccctg gtcctgctgg tccccgaggg 2220 gccaacggtg ctcccggcaa cgatggtgct aagggtgatg ctggtgcccc tggagctccc 2280 ggtagccagg gcgcccctgg ccttcaggga atgcctggtg aacgtggtgc agctggtctt 2340 ccagggccta agggtgacag aggtgatgct ggtcccaaag gtgctgatgg ctctcctggc 2400 aaagatggcg tccgtggtct gactggcccc attggtcctc ctggccctgc tggtgcccct 2460 ggtgacaagg gtgaaagtgg tcccagcggc cctgctggtc ccactggagc tcgtggtgcc 2520 cccggagacc gtggtgagcc tggtcccccc ggccctgctg gctttgctgg cccccctggt 2580 gctgacggcc aacctggtgc taaaggcgaa cctggtgatg ctggtgctaa aggcgatgct 2640 ggtccccctg gccctgccgg acccgctgga ccccctggcc ccattggtaa tgttggtgct 2700 cctggagcca aaggtgctcg cggcagcgct ggtccccctg gtgctactgg tttccctggt 2760 gctgctggcc gagtcggtcc tcctggcccc tctggaaatg ctggaccccc tggccctcct 2820 ggtcctgctg gcaaagaagg cggcaaaggt ccccgtggtg agactggccc tgctggacgt 2880 cctggtgaag ttggtccccc tggtccccct ggccctgctg gcgagaaagg atcccctggt 2940 gctgatggtc ctgctggtgc tcctggtact cccgggcctc aaggtattgc tggacagcgt 3000 ggtgtggtcg gcctgcctgg tcagagagga gagagaggct tccctggtct tcctggcccc 3060 tctggtgaac ctggcaaaca aggtccctct ggagcaagtg gtgaacgtgg tccccctggt 3120 cccatgggcc cccctggatt ggctggaccc cctggtgaat ctggacgtga gggggctcct 3180 ggtgccgaag gttcccctgg acgagacggt tctcctggcg ccaagggtga ccgtggtgag 3240 accggccccg ctggaccccc tggtgctcct ggtgctcctg gtgcccctgg ccccgttggc 3300 cctgctggca agagtggtga tcgtggtgag actggtcctg ctggtcccgc cggtcctgtc 3360 ggccctgttg gcgcccgtgg ccccgccgga ccccaaggcc cccgtggtga caagggtgag 3420 acaggcgaac agggcgacag aggcataaag ggtcaccgtg gcttctctgg cctccagggt 3480 ccccctggcc ctcctggctc tcctggtgaa caaggtccct ctggagcctc tggtcctgct 3540 ggtccccgag gtccccctgg ctctgctggt gctcctggca aagatggact caacggtctc 3600 cctggcccca ttgggccccc tggtcctcgc ggtcgcactg gtgatgctgg tcctgttggt 3660 ccccccggcc ctcctggacc tcctggtccc cctggtcctc ccagcgctgg tttcgacttc 3720 agcttcctgc cccagccacc tcaagagaag gctcacgatg gtggccgcta ctaccgggct 3780 gatgatgcca atgtggttcg tgaccgtgac ctcgaggtgg acaccaccct caagagcctg 3840 agccagcaga tcgagaacat ccggagccca gagggcagcc gcaagaaccc cgcccgcacc 3900 tgccgtgacc tcaagatgtg ccactctgac tggaagagtg gagagtactg gattgacccc 3960 aaccaaggct gcaacctgga tgccatcaaa gtcttctgca acatggagac tggtgagacc 4020 tgcgtgtacc ccactcagcc cagtgtggcc cagaagaact ggtacatcag caagaacccc 4080 aaggacaaga ggcatgtctg gttcggcgag agcatgaccg atggattcca gttcgagtat 4140 ggcggccagg gctccgaccc tgccgatgtg gccatccagc tgaccttcct gcgcctgatg 4200 tccaccgagg cctcccagaa catcacctac cactgcaaga acagcgtggc ctacatggac 4260 cagcagactg gcaacctcaa gaaggccctg ctcctccagg gctccaacga gatcgagatc 4320 cgcgccgagg gcaacagccg cttcacctac agcgtcactg tcgatggctg cacgagtcac 4380 accggagcct ggggcaagac agtgattgaa tacaaaacca ccaagacctc ccgcctgccc 4440 atcatcgatg tggccccctt ggacgttggt gccccagacc aggaattcgg cttcgacgtt 4500 ggccctgtct gcttcctgta aactccctcc atcccaacct ggctccctcc cacccaacca 4560 actttccccc caacccggaa acagacaagc aacccaaact gaaccccctc aaaagccaaa 4620 aaatgggaga caatttcaca tggactttgg aaaatatttt tttcctttgc attcatctct 4680 caaacttagt ttttatcttt gaccaaccga acatgaccaa aaaccaaaag tgcattcaac 4740 cttaccaaaa aaaaaaaaaa aaaaagaata aataaataac tttttaaaaa aggaagcttg 4800 gtccacttgc ttgaagaccc atgcgggggt aagtcccttt ctgcccgttg ggcttatgaa 4860 accccaatgc tgccctttct gctcctttct ccacaccccc cttggggcct cccctccact 4920 ccttcccaaa tctgtctccc cagaagacac aggaaacaat gtattgtctg cccagcaatc 4980 aaaggcaatg ctcaaacacc caagtggccc ccaccctcag cccgctcctg cccgcccagc 5040 acccccaggc cctgggggac ctggggttct cagactgcca aagaagcctt gccatctggc 5100 gctcccatgg ctcttgcaac atctcccctt cgtttttgag ggggtcatgc cgggggagcc 5160 accagcccct cactgggttc ggaggagagt caggaagggc cacgacaaag cagaaacatc 5220 ggatttgggg aacgcgtgtc aatcccttgt gccgcagggc tgggcgggag agactgttct 5280 gttccttgtg taactgtgtt gctgaaagac tacctcgttc ttgtcttgat gtgtcaccgg 5340 ggcaactgcc tgggggcggg gatgggggca gggtggaagc ggctccccat tttataccaa 5400 aggtgctaca tctatgtgat gggtggggtg gggagggaat cactggtgct atagaaattg 5460 agatgccccc ccaggccagc aaatgttcct ttttgttcaa agtctatttt tattccttga 5520 tatttttctt tttttttttt tttttttgtg gatggggact tgtgaatttt tctaaaggtg 5580 ctatttaaca tgggaggaga gcgtgtgcgg ctccagccca gcccgctgct cactttccac 5640 cctctctcca cctgcctctg gcttctcagg cctctgctct ccgacctctc tcctctgaaa 5700 ccctcctcca cagctgcagc ccatcctccc ggctccctcc tagtctgtcc tgcgtcctct 5760 gtccccgggt ttcagagaca acttcccaaa gcacaaagca gtttttcccc ctaggggtgg 5820 gaggaagcaa aagactctgt acctattttg tatgtgtata ataatttgag atgtttttaa 5880 ttattttgat tgctggaata aagcatgtgg aaatgaccca aacataa 5927 <210> 33 <211> 5411 <212> DNA <213> Homo sapiens <400> 33 gtgtcccata gtgtttccaa acttggaaag ggcgggggag ggcgggagga tgcggagggc 60 ggaggtatgc agacaacgag tcagagtttc cccttgaaag cctcaaaagt gtccacgtcc 120 tcaaaaagaa tggaaccaat ttaagaagcc agccccgtgg ccacgtccct tcccccattc 180 gctccctcct ctgcgccccc gcaggctcct cccagctgtg gctgcccggg cccccagccc 240 cagccctccc attggtggag gcccttttgg aggcacccta gggccaggga aacttttgcc 300 gtataaatag ggcagatccg ggctttatta ttttagcacc acggcagcag gaggtttcgg 360 ctaagttgga ggtactggcc acgactgcat gcccgcgccc gccaggtgat acctccgccg 420 gtgacccagg ggctctgcga cacaaggagt ctgcatgtct aagtgctaga catgctcagc 480 tttgtggata cgcggacttt gttgctgctt gcagtaacct tatgcctagc aacatgccaa 540 tctttacaag aggaaactgt aagaaagggc ccagccggag atagaggacc acgtggagaa 600 aggggtccac caggcccccc aggcagagat ggtgaagatg gtcccacagg ccctcctggt 660 ccacctggtc ctcctggccc ccctggtctc ggtgggaact ttgctgctca gtatgatgga 720 aaaggagttg gacttggccc tggaccaatg ggcttaatgg gacctagagg cccacctggt 780 gcagctggag ccccaggccc tcaaggtttc caaggacctg ctggtgagcc tggtgaacct 840 ggtcaaactg gtcctgcagg tgctcgtggt ccagctggcc ctcctggcaa ggctggtgaa 900 gatggtcacc ctggaaaacc cggacgacct ggtgagagag gagttgttgg accacagggt 960 gctcgtggtt tccctggaac tcctggactt cctggcttca aaggcattag gggacacaat 1020 ggtctggatg gattgaaggg acagcccggt gctcctggtg tgaagggtga acctggtgcc 1080 cctggtgaaa atggaactcc aggtcaaaca ggagcccgtg ggcttcctgg tgagagagga 1140 cgtgttggtg cccctggccc agctggtgcc cgtggcagtg atggaagtgt gggtcccgtg 1200 ggtcctgctg gtcccattgg gtctgctggc cctccaggct tcccaggtgc ccctggcccc 1260 aagggtgaaa ttggagctgt tggtaacgct ggtcctgctg gtcccgccgg tccccgtggt 1320 gaagtgggtc ttccaggcct ctccggcccc gttggacctc ctggtaatcc tggagcaaac 1380 ggccttactg gtgccaaggg tgctgctggc cttcccggcg ttgctggggc tcccggcctc 1440 cctggacccc gcggtattcc tggccctgtt ggtgctgccg gtgctactgg tgccagagga 1500 cttgttggtg agcctggtcc agctggctcc aaaggagaga gcggtaacaa gggtgagccc 1560 ggctctgctg ggccccaagg tcctcctggt cccagtggtg aagaaggaaa gagaggccct 1620 aatggggaag ctggatctgc cggccctcca ggacctcctg ggctgagagg tagtcctggt 1680 tctcgtggtc ttcctggagc tgatggcaga gctggcgtca tgggccctcc tggtagtcgt 1740 ggtgcaagtg gccctgctgg agtccgagga cctaatggag atgctggtcg ccctggggag 1800 cctggtctca tgggacccag aggtcttcct ggttcccctg gaaatatcgg ccccgctgga 1860 aaagaaggtc ctgtcggcct ccctggcatc gacggcaggc ctggcccaat tggcccagct 1920 ggagcaagag gagagcctgg caacattgga ttccctggac ccaaaggccc cactggtgat 1980 cctggcaaaa acggtgataa aggtcatgct ggtcttgctg gtgctcgggg tgctccaggt 2040 cctgatggaa acaatggtgc tcagggacct cctggaccac agggtgttca aggtggaaaa 2100 ggtgaacagg gtccccctgg tcctccaggc ttccagggtc tgcctggccc ctcaggtccc 2160 gctggtgaag ttggcaaacc aggagaaagg ggtctccatg gtgagtttgg tctccctggt 2220 cctgctggtc caagagggga acgcggtccc ccaggtgaga gtggtgctgc cggtcctact 2280 ggtcctattg gaagccgagg tccttctgga cccccagggc ctgatggaaa caagggtgaa 2340 cctggtgtgg ttggtgctgt gggcactgct ggtccatctg gtcctagtgg actcccagga 2400 gagaggggtg ctgctggcat acctggaggc aagggagaaa agggtgaacc tggtctcaga 2460 ggtgaaattg gtaaccctgg cagagatggt gctcgtggtg ctcctggtgc tgtaggtgcc 2520 cctggtcctg ctggagccac aggtgaccgg ggcgaagctg gggctgctgg tcctgctggt 2580 cctgctggtc ctcggggaag ccctggtgaa cgtggtgagg tcggtcctgc tggccccaat 2640 ggatttgctg gtcctgctgg tgctgctggt caacctggtg ctaaaggaga aagaggagcc 2700 aaagggccta agggtgaaaa cggtgttgtt ggtcccacag gccccgttgg agctgctggc 2760 ccagctggtc caaatggtcc ccccggtcct gctggaagtc gtggtgatgg aggcccccct 2820 ggtatgactg gtttccctgg tgctgctgga cggactggtc ccccaggacc ctctggtatt 2880 tctggccctc ctggtccccc tggtcctgct gggaaagaag ggcttcgtgg tcctcgtggt 2940 gaccaaggtc cagttggccg aactggagaa gtaggtgcag ttggtccccc tggcttcgct 3000 ggtgagaagg gtccctctgg agaggctggt actgctggac ctcctggcac tccaggtcct 3060 cagggtcttc ttggtgctcc tggtattctg ggtctccctg gctcgagagg tgaacgtggt 3120 ctaccaggtg ttgctggtgc tgtgggtgaa cctggtcctc ttggcattgc cggccctcct 3180 ggggcccgtg gtcctcctgg tgctgtgggt agtcctggag tcaacggtgc tcctggtgaa 3240 gctggtcgtg atggcaaccc tgggaacgat ggtcccccag gtcgcgatgg tcaacccgga 3300 cacaagggag agcgcggtta ccctggcaat attggtcccg ttggtgctgc aggtgcacct 3360 ggtcctcatg gccccgtggg tcctgctggc aaacatggaa accgtggtga aactggtcct 3420 tctggtcctg ttggtcctgc tggtgctgtt ggcccaagag gtcctagtgg cccacaaggc 3480 attcgtggcg ataagggaga gcccggtgaa aaggggccca gaggtcttcc tggcttaaag 3540 ggacacaatg gattgcaagg tctgcctggt atcgctggtc accatggtga tcaaggtgct 3600 cctggctccg tgggtcctgc tggtcctagg ggccctgctg gtccttctgg ccctgctgga 3660 aaagatggtc gcactggaca tcctggtaca gttggacctg ctggcattcg aggccctcag 3720 ggtcaccaag gccctgctgg cccccctggt ccccctggcc ctcctggacc tccaggtgta 3780 agcggtggtg gttatgactt tggttacgat ggagacttct acagggctga ccagcctcgc 3840 tcagcacctt ctctcagacc caaggactat gaagttgatg ctactctgaa gtctctcaac 3900 aaccagattg agacccttct tactcctgaa ggctctagaa agaacccagc tcgcacatgc 3960 cgtgacttga gactcagcca cccagagtgg agcagtggtt actactggat tgaccctaac 4020 caaggatgca ctatggatgc tatcaaagta tactgtgatt tctctactgg cgaaacctgt 4080 atccgggccc aacctgaaaa catcccagcc aagaactggt ataggagctc caaggacaag 4140 aaacacgtct ggctaggaga aactatcaat gctggcagcc agtttgaata taatgtagaa 4200 ggagtgactt ccaaggaaat ggctacccaa cttgccttca tgcgcctgct ggccaactat 4260 gcctctcaga acatcaccta ccactgcaag aacagcattg catacatgga tgaggagact 4320 ggcaacctga aaaaggctgt cattctacag ggctctaatg atgttgaact tgttgctgag 4380 ggcaacagca ggttcactta cactgttctt gtagatggct gctctaaaaa gacaaatgaa 4440 tggggaaaga caatcattga atacaaaaca aataagccat cacgcctgcc cttccttgat 4500 attgcacctt tggacatcgg tggtgctgac caggaattct ttgtggacat tggcccagtc 4560 tgtttcaaat aaatgaactc aatctaaatt aaaaaagaaa gaaatttgaa aaaactttct 4620 ctttgccatt tcttcttctt cttttttaac tgaaagctga atccttccat ttcttctgca 4680 catctacttg cttaaattgt gggcaaaaga gaaaaagaag gattgatcag agcattgtgc 4740 aatacagttt cattaactcc ttcccccgct cccccaaaaa tttgaatttt tttttcaaca 4800 ctcttacacc tgttatggaa aatgtcaacc tttgtaagaa aaccaaaata aaaattgaaa 4860 aataaaaacc ataaacattt gcaccacttg tggcttttga atatcttcca cagagggaag 4920 tttaaaaccc aaacttccaa aggtttaaac tacctcaaaa cactttccca tgagtgtgat 4980 ccacattgtt aggtgctgac ctagacagag atgaactgag gtccttgttt tgttttgttc 5040 ataatacaaa ggtgctaatt aatagtattt cagatacttg aagaatgttg atggtgctag 5100 aagaatttga gaagaaatac tcctgtattg agttgtatcg tgtggtgtat tttttaaaaa 5160 atttgattta gcattcatat tttccatctt attcccaatt aaaagtatgc agattatttg 5220 cccaaatctt cttcagattc agcatttgtt ctttgccagt ctcattttca tcttcttcca 5280 tggttccaca gaagctttgt ttcttgggca agcagaaaaa ttaaattgta cctattttgt 5340 atatgtgaga tgtttaaata aattgtgaaa aaaatgaaat aaagcatgtt tggttttcca 5400 aaagaacata t 5411 <210> 34 <211> 1633 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vascular signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Prolyl 4-hydroxylase beta subunit and flanking regions <400> 34 ctcgagtaaa ccatggctca tgctagggtt ttgcttttgg ctcttgctgt tcttgctact 60 gctgctgttg ctgtggcttc ttcttcatct ttcgctgatt ctaacccaat taggccagtg 120 actgatagag ctgcttctac tcttgctcaa ttggtcgaca tggatgctcc agaagaggag 180 gatcacgttc ttgtgcttag gaagtctaac ttcgctgaag ctcttgctgc tcacaagtac 240 cttcttgtgg agttttatgc tccttggtgc ggacattgca aagctcttgc tccagagtat 300 gctaaggctg ctggaaagtt gaaggctgag ggatctgaaa ttaggcttgc taaagtggat 360 gctactgagg agtctgatct tgctcaacag tacggagtta ggggataccc aactattaag 420 ttcttcagga acggagatac tgcttctcca aaggagtata ctgctggaag ggaggctgat 480 gatattgtga actggcttaa gaagagaact ggaccagctg ctactactct tccagatgga 540 gctgctgctg aatctcttgt ggagtcatct gaggtggcag tgattggatt cttcaaggat 600 gtggagtctg attctgctaa gcagttcctt caagctgctg aggctattga tgatattcca 660 ttcggaatta cttctaactc tgatgtgttc tctaagtacc agcttgataa ggatggagtg 720 gtgcttttca agaaattcga tgagggaagg aacaatttcg agggagaggt gacaaaggag 780 aaccttcttg atttcattaa gcacaaccag cttccacttg tgattgagtt cactgagcag 840 actgctccaa agattttcgg aggagagatt aagactcaca ttcttctttt ccttccaaag 900 tctgtgtctg attacgatgg aaagttgtct aacttcaaga ctgctgctga gtctttcaag 960 ggaaagattc ttttcatttt cattgattct gatcacactg ataaccagag gattcttgag 1020 ttcttcggac ttaagaagga agagtgccca gctgttaggc ttattactct tgaggaggag 1080 atgactaagt acaagccaga gtctgaagaa cttactgctg agaggattac tgagttctgc 1140 cacagattcc ttgagggaaa gattaagcca caccttatgt ctcaagagct tccagaggat 1200 tgggataagc agccagttaa ggtgttggtg ggtaaaaact tcgaggatgt ggctttcgat 1260 gagaagaaga acgtgttcgt ggagttctac gcaccttggt gtggtcactg taagcagctt 1320 gctccaattt gggataagtt gggagagact tacaaggatc acgagaacat tgtgattgct 1380 aagatggatt ctactgctaa cgaggtggag gctgttaagg ttcactcttt cccaactttg 1440 aagttcttcc cagcttctgc tgataggact gtgattgatt acaacggaga aaggactctt 1500 gatggattca agaagttcct tgagtctgga ggacaagatg gagctggaga tgatgatgat 1560 cttgaggatt tggaagaagc tgaggagcca gatatggagg aggatgatga tcagaaggct 1620 gtgtgatgag ctc 1633 <210> 35 <211> 1723 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vascular signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Prolyl 4-hydroxylase alpha-1 subunit and flanking regions <400> 35 ctcgagtaaa ccatggctca tgctagggtt ttgcttttgg ctcttgctgt tcttgctact 60 gctgctgttg ctgtggcttc ttcttcatct ttcgctgatt ctaacccaat taggccagtg 120 actgatagag ctgcttctac tcttgctcaa ttggtcgaca tgcacccagg attcttcact 180 tctattggac agatgactga tcttattcac actgagaagg atcttgtgac ttctcttaag 240 gattacatta aggctgagga ggataagttg gagcagatta agaagtgggc tgagaagttg 300 gataggctta cttctactgc tacaaaagat ccagagggat tcgttggtca tccagtgaac 360 gctttcaagt tgatgaagag gcttaacact gagtggagtg agcttgagaa ccttgtgctt 420 aaggatatgt ctgatggatt catttctaac cttactattc agaggcagta cttcccaaat 480 gatgaggatc aagtgggagc tgctaaggct cttcttaggc ttcaggatac ttacaacctt 540 gatactgata caatttctaa gggaaacctt ccaggagtta agcacaagtc tttccttact 600 gctgaggatt gcttcgagct tggaaaggtt gcatacactg aggctgatta ctaccacact 660 gagctttgga tggaacaagc tcttaggcaa cttgatgagg gagagatttc tactattgat 720 aaggtgtcag tgcttgatta cctttcttac gctgtgtacc agcagggtga tcttgataag 780 gctcttttgc ttactaagaa gttgcttgag cttgatccag aacatcagag ggctaacgga 840 aaccttaagt acttcgagta cattatggct aaggaaaagg atgtgaacaa gtctgcttct 900 gatgatcagt ctgatcaaaa gactactcca aagaagaagg gagtggctgt tgattatctt 960 cctgagaggc agaagtatga gatgttgtgt aggggagagg gtattaagat gactccaagg 1020 aggcagaaga agttgttctg caggtatcac gatggaaaca ggaacccaaa gttcattctt 1080 gctccagcta agcaagaaga tgagtgggat aagccaagga ttattaggtt ccacgatatt 1140 atttctgatg ctgagattga gattgtgaag gatcttgcta agccaagact taggagggct 1200 actatttcta accctattac tggtgatctt gagactgtgc actacaggat ttctaagtct 1260 gcttggcttt ctggatacga gaacccagtg gtgtctagga ttaacatgag gattcaggat 1320 cttactggac ttgatgtgtc tactgctgag gagcttcaag ttgctaacta cggagttgga 1380 ggacaatatg agccacactt cgatttcgct aggaaggatg agccagatgc ttttaaggag 1440 cttggaactg gaaacaggat tgctacttgg cttttctaca tgtctgatgt ttctgctgga 1500 ggagctactg ttttcccaga agtgggagct tctgtttggc caaagaaggg aactgctgtg 1560 ttctggtaca accttttcgc ttctggagag ggagattact ctactaggca tgctgcttgc 1620 ccagttcttg ttggaaacaa gtgggtgtca aacaagtggc ttcatgagag gggacaagag 1680 tttagaaggc catgcactct ttctgagctt gagtgatgag ctc 1723 <210> 36 <211> 1489 <212> PRT <213> Homo sapiens <400> 36 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Gln 35 40 45 Glu Glu Gly Gln Val Glu Gly Gln Asp Glu Asp Ile Pro Pro Ile Thr 50 55 60 Cys Val Gln Asn Gly Leu Arg Tyr His Asp Arg Asp Val Trp Lys Pro 65 70 75 80 Glu Pro Cys Arg Ile Cys Val Cys Asp Asn Gly Lys Val Leu Cys Asp 85 90 95 Asp Val Ile Cys Asp Glu Thr Lys Asn Cys Pro Gly Ala Glu Val Pro 100 105 110 Glu Gly Glu Cys Cys Pro Val Cys Pro Asp Gly Ser Glu Ser Pro Thr 115 120 125 Asp Gln Glu Thr Thr Gly Val Glu Gly Pro Lys Gly Asp Thr Gly Pro 130 135 140 Arg Gly Pro Arg Gly Pro Ala Gly Pro Pro Gly Arg Asp Gly Ile Pro 145 150 155 160 Gly Gln Pro Gly Leu Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 165 170 175 Pro Pro Gly Leu Gly Gly Asn Phe Ala Pro Gln Leu Ser Tyr Gly Tyr 180 185 190 Asp Glu Lys Ser Thr Gly Gly Ile Ser Val Pro Gly Pro Met Gly Pro 195 200 205 Ser Gly Pro Arg Gly Leu Pro Gly Pro Pro Gly Ala Pro Gly Pro Gln 210 215 220 Gly Phe Gln Gly Pro Pro Gly Glu Pro Gly Glu Pro Gly Ala Ser Gly 225 230 235 240 Pro Met Gly Pro Arg Gly Pro Pro Gly Pro Pro Gly Lys Asn Gly Asp 245 250 255 Asp Gly Glu Ala Gly Lys Pro Gly Arg Pro Gly Glu Arg Gly Pro Pro 260 265 270 Gly Pro Gln Gly Ala Arg Gly Leu Pro Gly Thr Ala Gly Leu Pro Gly 275 280 285 Met Lys Gly His Arg Gly Phe Ser Gly Leu Asp Gly Ala Lys Gly Asp 290 295 300 Ala Gly Pro Ala Gly Pro Lys Gly Glu Pro Gly Ser Pro Gly Glu Asn 305 310 315 320 Gly Ala Pro Gly Gln Met Gly Pro Arg Gly Leu Pro Gly Glu Arg Gly 325 330 335 Arg Pro Gly Ala Pro Gly Pro Ala Gly Ala Arg Gly Asn Asp Gly Ala 340 345 350 Thr Gly Ala Ala Gly Pro Pro Gly Pro Thr Gly Pro Ala Gly Pro Pro 355 360 365 Gly Phe Pro Gly Ala Val Gly Ala Lys Gly Glu Ala Gly Pro Gln Gly 370 375 380 Pro Arg Gly Ser Glu Gly Pro Gln Gly Val Arg Gly Glu Pro Gly Pro 385 390 395 400 Pro Gly Pro Ala Gly Ala Ala Gly Pro Ala Gly Asn Pro Gly Ala Asp 405 410 415 Gly Gln Pro Gly Ala Lys Gly Ala Asn Gly Ala Pro Gly Ile Ala Gly 420 425 430 Ala Pro Gly Phe Pro Gly Ala Arg Gly Pro Ser Gly Pro Gln Gly Pro 435 440 445 Gly Gly Pro Pro Gly Pro Lys Gly Asn Ser Gly Glu Pro Gly Ala Pro 450 455 460 Gly Ser Lys Gly Asp Thr Gly Ala Lys Gly Glu Pro Gly Pro Val Gly 465 470 475 480 Val Gln Gly Pro Pro Gly Pro Ala Gly Glu Glu Gly Lys Arg Gly Ala 485 490 495 Arg Gly Glu Pro Gly Pro Thr Gly Leu Pro Gly Pro Pro Gly Glu Arg 500 505 510 Gly Gly Pro Gly Ser Arg Gly Phe Pro Gly Ala Asp Gly Val Ala Gly 515 520 525 Pro Lys Gly Pro Ala Gly Glu Arg Gly Ser Pro Gly Pro Ala Gly Pro 530 535 540 Lys Gly Ser Pro Gly Glu Ala Gly Arg Pro Gly Glu Ala Gly Leu Pro 545 550 555 560 Gly Ala Lys Gly Leu Thr Gly Ser Pro Gly Ser Pro Gly Pro Asp Gly 565 570 575 Lys Thr Gly Pro Pro Gly Pro Ala Gly Gln Asp Gly Arg Pro Gly Pro 580 585 590 Pro Gly Pro Pro Gly Ala Arg Gly Gln Ala Gly Val Met Gly Phe Pro 595 600 605 Gly Pro Lys Gly Ala Ala Gly Glu Pro Gly Lys Ala Gly Glu Arg Gly 610 615 620 Val Pro Gly Pro Pro Gly Ala Val Gly Pro Ala Gly Lys Asp Gly Glu 625 630 635 640 Ala Gly Ala Gln Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly Glu Arg 645 650 655 Gly Glu Gln Gly Pro Ala Gly Ser Pro Gly Phe Gln Gly Leu Pro Gly 660 665 670 Pro Ala Gly Pro Pro Gly Glu Ala Gly Lys Pro Gly Glu Gln Gly Val 675 680 685 Pro Gly Asp Leu Gly Ala Pro Gly Pro Ser Gly Ala Arg Gly Glu Arg 690 695 700 Gly Phe Pro Gly Glu Arg Gly Val Gln Gly Pro Pro Gly Pro Ala Gly 705 710 715 720 Pro Arg Gly Ala Asn Gly Ala Pro Gly Asn Asp Gly Ala Lys Gly Asp 725 730 735 Ala Gly Ala Pro Gly Ala Pro Gly Ser Gln Gly Ala Pro Gly Leu Gln 740 745 750 Gly Met Pro Gly Glu Arg Gly Ala Ala Gly Leu Pro Gly Pro Lys Gly 755 760 765 Asp Arg Gly Asp Ala Gly Pro Lys Gly Ala Asp Gly Ser Pro Gly Lys 770 775 780 Asp Gly Val Arg Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala 785 790 795 800 Gly Ala Pro Gly Asp Lys Gly Glu Ser Gly Pro Ser Gly Pro Ala Gly 805 810 815 Pro Thr Gly Ala Arg Gly Ala Pro Gly Asp Arg Gly Glu Pro Gly Pro 820 825 830 Pro Gly Pro Ala Gly Phe Ala Gly Pro Pro Gly Ala Asp Gly Gln Pro 835 840 845 Gly Ala Lys Gly Glu Pro Gly Asp Ala Gly Ala Lys Gly Asp Ala Gly 850 855 860 Pro Pro Gly Pro Ala Gly Pro Ala Gly Pro Pro Gly Pro Ile Gly Asn 865 870 875 880 Val Gly Ala Pro Gly Ala Lys Gly Ala Arg Gly Ser Ala Gly Pro Pro 885 890 895 Gly Ala Thr Gly Phe Pro Gly Ala Ala Gly Arg Val Gly Pro Pro Gly 900 905 910 Pro Ser Gly Asn Ala Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Lys 915 920 925 Glu Gly Gly Lys Gly Pro Arg Gly Glu Thr Gly Pro Ala Gly Arg Pro 930 935 940 Gly Glu Val Gly Pro Pro Gly Pro Pro Gly Pro Ala Gly Glu Lys Gly 945 950 955 960 Ser Pro Gly Ala Asp Gly Pro Ala Gly Ala Pro Gly Thr Pro Gly Pro 965 970 975 Gln Gly Ile Ala Gly Gln Arg Gly Val Val Gly Leu Pro Gly Gln Arg 980 985 990 Gly Glu Arg Gly Phe Pro Gly Leu Pro Gly Pro Ser Gly Glu Pro Gly 995 1000 1005 Lys Gln Gly Pro Ser Gly Ala Ser Gly Glu Arg Gly Pro Pro Gly 1010 1015 1020 Pro Met Gly Pro Pro Gly Leu Ala Gly Pro Pro Gly Glu Ser Gly 1025 1030 1035 Arg Glu Gly Ala Pro Gly Ala Glu Gly Ser Pro Gly Arg Asp Gly 1040 1045 1050 Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly 1055 1060 1065 Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro Val Gly 1070 1075 1080 Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala Gly 1085 1090 1095 Pro Ala Gly Pro Val Gly Pro Ala Gly Ala Arg Gly Pro Ala Gly 1100 1105 1110 Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly 1115 1120 1125 Asp Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly 1130 1135 1140 Pro Pro Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly 1145 1150 1155 Ala Ser Gly Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly 1160 1165 1170 Ala Pro Gly Lys Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly 1175 1180 1185 Pro Pro Gly Pro Arg Gly Arg Thr Gly Asp Ala Gly Pro Val Gly 1190 1195 1200 Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Ser 1205 1210 1215 Ala Gly Phe Asp Phe Ser Phe Leu Pro Gln Pro Pro Gln Glu Lys 1220 1225 1230 Ala His Asp Gly Gly Arg Tyr Tyr Arg Ala Asp Asp Ala Asn Val 1235 1240 1245 Val Arg Asp Arg Asp Leu Glu Val Asp Thr Thr Leu Lys Ser Leu 1250 1255 1260 Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro Glu Gly Ser Arg Lys 1265 1270 1275 Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met Cys His Ser Asp 1280 1285 1290 Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln Gly Cys Asn 1295 1300 1305 Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly Glu Thr 1310 1315 1320 Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp Tyr 1325 1330 1335 Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu 1340 1345 1350 Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser 1355 1360 1365 Asp Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met 1370 1375 1380 Ser Thr Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser 1385 1390 1395 Val Ala Tyr Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu 1400 1405 1410 Leu Leu Lys Gly Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn 1415 1420 1425 Ser Arg Phe Thr Tyr Ser Val Thr Val Asp Gly Cys Thr Ser His 1430 1435 1440 Thr Gly Ala Trp Gly Lys Thr Val Ile Glu Tyr Lys Thr Thr Lys 1445 1450 1455 Thr Ser Arg Leu Pro Ile Ile Asp Val Ala Pro Leu Asp Val Gly 1460 1465 1470 Ala Pro Asp Gln Glu Phe Gly Phe Asp Val Gly Pro Val Cys Phe 1475 1480 1485 Leu <210> 37 <211> 1389 <212> PRT <213> Homo sapiens <400> 37 Met Ala His Ala Arg Val Leu Leu Leu Ala Leu Ala Val Leu Ala Thr 1 5 10 15 Ala Ala Val Ala Val Ala Ser Ser Ser Ser Phe Ala Asp Ser Asn Pro 20 25 30 Ile Arg Pro Val Thr Asp Arg Ala Ala Ser Thr Leu Ala Gln Leu Leu 35 40 45 Gln Glu Glu Thr Val Arg Lys Gly Pro Ala Gly Asp Arg Gly Pro Arg 50 55 60 Gly Glu Arg Gly Pro Pro Gly Pro Pro Gly Arg Asp Gly Glu Asp Gly 65 70 75 80 Pro Thr Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu 85 90 95 Gly Gly Asn Phe Ala Ala Gln Tyr Asp Gly Lys Gly Val Gly Leu Gly 100 105 110 Pro Gly Pro Met Gly Leu Met Gly Pro Arg Gly Pro Pro Gly Ala Ala 115 120 125 Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Ala Gly Glu Pro Gly 130 135 140 Glu Pro Gly Gln Thr Gly Pro Ala Gly Ala Arg Gly Pro Ala Gly Pro 145 150 155 160 Pro Gly Lys Ala Gly Glu Asp Gly His Pro Gly Lys Pro Gly Arg Pro 165 170 175 Gly Glu Arg Gly Val Val Gly Pro Gln Gly Ala Arg Gly Phe Pro Gly 180 185 190 Thr Pro Gly Leu Pro Gly Phe Lys Gly Ile Arg Gly His Asn Gly Leu 195 200 205 Asp Gly Leu Lys Gly Gln Pro Gly Ala Pro Gly Val Lys Gly Glu Pro 210 215 220 Gly Ala Pro Gly Glu Asn Gly Thr Pro Gly Gln Thr Gly Ala Arg Gly 225 230 235 240 Leu Pro Gly Glu Arg Gly Arg Val Gly Ala Pro Gly Pro Ala Gly Ala 245 250 255 Arg Gly Ser Asp Gly Ser Val Gly Pro Val Gly Pro Ala Gly Pro Ile 260 265 270 Gly Ser Ala Gly Pro Pro Gly Phe Pro Gly Ala Pro Gly Pro Lys Gly 275 280 285 Glu Ile Gly Ala Val Gly Asn Ala Gly Pro Thr Gly Pro Ala Gly Pro 290 295 300 Arg Gly Glu Val Gly Leu Pro Gly Leu Ser Gly Pro Val Gly Pro Pro 305 310 315 320 Gly Asn Pro Gly Ala Asn Gly Leu Thr Gly Ala Lys Gly Ala Ala Gly 325 330 335 Leu Pro Gly Val Ala Gly Ala Pro Gly Leu Pro Gly Pro Arg Gly Ile 340 345 350 Pro Gly Pro Val Gly Ala Ala Gly Ala Thr Gly Ala Arg Gly Leu Val 355 360 365 Gly Glu Pro Gly Pro Ala Gly Ser Lys Gly Glu Ser Gly Asn Lys Gly 370 375 380 Glu Pro Gly Ser Ala Gly Pro Gln Gly Pro Pro Gly Pro Ser Gly Glu 385 390 395 400 Glu Gly Lys Arg Gly Pro Asn Gly Glu Ala Gly Ser Ala Gly Pro Pro 405 410 415 Gly Pro Pro Gly Leu Arg Gly Ser Pro Gly Ser Arg Gly Leu Pro Gly 420 425 430 Ala Asp Gly Arg Ala Gly Val Met Gly Pro Pro Gly Ser Arg Gly Ala 435 440 445 Ser Gly Pro Ala Gly Val Arg Gly Pro Asn Gly Asp Ala Gly Arg Pro 450 455 460 Gly Glu Pro Gly Leu Met Gly Pro Arg Gly Leu Pro Gly Ser Pro Gly 465 470 475 480 Asn Ile Gly Pro Ala Gly Lys Glu Gly Pro Val Gly Leu Pro Gly Ile 485 490 495 Asp Gly Arg Pro Gly Pro Ile Gly Pro Ala Gly Ala Arg Gly Glu Pro 500 505 510 Gly Asn Ile Gly Phe Pro Gly Pro Lys Gly Pro Thr Gly Asp Pro Gly 515 520 525 Lys Asn Gly Asp Lys Gly His Ala Gly Leu Ala Gly Ala Arg Gly Ala 530 535 540 Pro Gly Pro Asp Gly Asn Asn Gly Ala Gln Gly Pro Pro Gly Pro Gln 545 550 555 560 Gly Val Gln Gly Gly Lys Gly Glu Gln Gly Pro Ala Gly Pro Pro Gly 565 570 575 Phe Gln Gly Leu Pro Gly Pro Ser Gly Pro Ala Gly Glu Val Gly Lys 580 585 590 Pro Gly Glu Arg Gly Leu His Gly Glu Phe Gly Leu Pro Gly Pro Ala 595 600 605 Gly Pro Arg Gly Glu Arg Gly Pro Pro Gly Glu Ser Gly Ala Ala Gly 610 615 620 Pro Thr Gly Pro Ile Gly Ser Arg Gly Pro Ser Gly Pro Pro Gly Pro 625 630 635 640 Asp Gly Asn Lys Gly Glu Pro Gly Val Val Gly Ala Val Gly Thr Ala 645 650 655 Gly Pro Ser Gly Pro Ser Gly Leu Pro Gly Glu Arg Gly Ala Ala Gly 660 665 670 Ile Pro Gly Gly Lys Gly Glu Lys Gly Glu Pro Gly Leu Arg Gly Glu 675 680 685 Ile Gly Asn Pro Gly Arg Asp Gly Ala Arg Gly Ala His Gly Ala Val 690 695 700 Gly Ala Pro Gly Pro Ala Gly Ala Thr Gly Asp Arg Gly Glu Ala Gly 705 710 715 720 Ala Ala Gly Pro Ala Gly Pro Ala Gly Pro Arg Gly Ser Pro Gly Glu 725 730 735 Arg Gly Glu Val Gly Pro Ala Gly Pro Asn Gly Phe Ala Gly Pro Ala 740 745 750 Gly Ala Ala Gly Gln Pro Gly Ala Lys Gly Glu Arg Gly Gly Lys Gly 755 760 765 Pro Lys Gly Glu Asn Gly Val Val Gly Pro Thr Gly Pro Val Gly Ala 770 775 780 Ala Gly Pro Ala Gly Pro Asn Gly Pro Pro Gly Pro Ala Gly Ser Arg 785 790 795 800 Gly Asp Gly Gly Pro Pro Gly Met Thr Gly Phe Pro Gly Ala Ala Gly 805 810 815 Arg Thr Gly Pro Pro Gly Pro Ser Gly Ile Ser Gly Pro Pro Gly Pro 820 825 830 Pro Gly Pro Ala Gly Lys Glu Gly Leu Arg Gly Pro Arg Gly Asp Gln 835 840 845 Gly Pro Val Gly Arg Thr Gly Glu Val Gly Ala Val Gly Pro Pro Gly 850 855 860 Phe Ala Gly Glu Lys Gly Pro Ser Gly Glu Ala Gly Thr Ala Gly Pro 865 870 875 880 Pro Gly Thr Pro Gly Pro Gln Gly Leu Leu Gly Ala Pro Gly Ile Leu 885 890 895 Gly Leu Pro Gly Ser Arg Gly Glu Arg Gly Leu Pro Gly Val Ala Gly 900 905 910 Ala Val Gly Glu Pro Gly Pro Leu Gly Ile Ala Gly Pro Pro Gly Ala 915 920 925 Arg Gly Pro Pro Gly Ala Val Gly Ser Pro Gly Val Asn Gly Ala Pro 930 935 940 Gly Glu Ala Gly Arg Asp Gly Asn Pro Gly Asn Asp Gly Pro Pro Gly 945 950 955 960 Arg Asp Gly Gln Pro Gly His Lys Gly Glu Arg Gly Tyr Pro Gly Asn 965 970 975 Ile Gly Pro Val Gly Ala Ala Gly Ala Pro Gly Pro His Gly Pro Val 980 985 990 Gly Pro Ala Gly Lys His Gly Asn Arg Gly Glu Thr Gly Pro Ser Gly 995 1000 1005 Pro Val Gly Pro Ala Gly Ala Val Gly Pro Arg Gly Pro Ser Gly 1010 1015 1020 Pro Gln Gly Ile Arg Gly Asp Lys Gly Glu Pro Gly Glu Lys Gly 1025 1030 1035 Pro Arg Gly Leu Pro Gly Phe Lys Gly His Asn Gly Leu Gln Gly 1040 1045 1050 Leu Pro Gly Ile Ala Gly His His Gly Asp Gln Gly Ala Pro Gly 1055 1060 1065 Ser Val Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly Pro Ser Gly 1070 1075 1080 Pro Ala Gly Lys Asp Gly Arg Thr Gly His Pro Gly Thr Val Gly 1085 1090 1095 Pro Ala Gly Ile Arg Gly Pro Gln Gly His Gln Gly Pro Ala Gly 1100 1105 1110 Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Val Ser Gly 1115 1120 1125 Gly Gly Tyr Asp Phe Gly Tyr Asp Gly Asp Phe Tyr Arg Ala Asp 1130 1135 1140 Gln Pro Arg Ser Ala Pro Ser Leu Arg Pro Lys Asp Tyr Glu Val 1145 1150 1155 Asp Ala Thr Leu Lys Ser Leu Asn Asn Gln Ile Glu Thr Leu Leu 1160 1165 1170 Thr Pro Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp 1175 1180 1185 Leu Arg Leu Ser His Pro Glu Trp Ser Ser Gly Tyr Tyr Trp Ile 1190 1195 1200 Asp Pro Asn Gln Gly Cys Thr Met Glu Ala Ile Lys Val Tyr Cys 1205 1210 1215 Asp Phe Pro Thr Gly Glu Thr Cys Ile Arg Ala Gln Pro Glu Asn 1220 1225 1230 Ile Pro Ala Lys Asn Trp Tyr Arg Ser Ser Lys Asp Lys Lys His 1235 1240 1245 Val Trp Leu Gly Glu Thr Ile Asn Ala Gly Ser Gln Phe Glu Tyr 1250 1255 1260 Asn Val Glu Gly Val Thr Ser Lys Glu Met Ala Thr Gln Leu Ala 1265 1270 1275 Phe Met Arg Leu Leu Ala Asn Tyr Ala Ser Gln Asn Ile Thr Tyr 1280 1285 1290 His Cys Lys Asn Ser Ile Ala Tyr Met Asp Glu Glu Thr Gly Asn 1295 1300 1305 Leu Lys Lys Ala Val Ile Leu Gln Gly Ser Asn Asp Val Glu Leu 1310 1315 1320 Val Ala Glu Gly Asn Ser Arg Phe Thr Tyr Thr Val Leu Val Asp 1325 1330 1335 Gly Cys Ser Lys Lys Thr Asn Glu Trp Gly Lys Thr Ile Ile Glu 1340 1345 1350 Tyr Lys Thr Asn Lys Pro Ser Arg Leu Pro Phe Leu Asp Ile Ala 1355 1360 1365 Pro Leu Asp Ile Gly Gly Ala Asp His Glu Phe Phe Val Asp Ile 1370 1375 1380 Gly Pro Val Cys Phe Lys 1385 <210> 38 <211> 2888 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the vascular signal sequence of barley gene for Thiol protease aleurain precursor fused to the human Lysyl hydroxylase 3 and flanking regions <400> 38 gcgaattcgc tagctatcac tgaaaagaca gcaagacaat ggtgtctcga tgcaccagaa 60 ccacatcttt gcagcagatg tgaagcagcc agagtggtcc acaagacgca ctcagaaaag 120 gcatcttcta ccgacacaga aaaagacaac cacagctcat catccaacat gtagactgtc 180 gttatgcgtc ggctgaagat aagactgacc ccaggccagc actaaagaag aaataatgca 240 agtggtccta gctccacttt agctttaata attatgtttc attattattc tctgcttttg 300 ctctctatat aaagagcttg tattttcatt tgaaggcaga ggcgaacaca cacacagaac 360 ctccctgctt acaaaccaga tcttaaacca tggctcacgc tagggttttg cttcttgctc 420 ttgctgttct tgctactgct gctgttgctg tggcttcttc aagttctttc gctgattcta 480 acccaattag gccagtgact gatagagctg cttctactct tgctcaattg agatctatgt 540 ctgatagacc aaggggaagg gatccagtta atccagagaa gttgcttgtg attactgtgg 600 ctactgctga gactgaagga taccttagat tccttaggag tgctgagttc ttcaactaca 660 ctgtgaggac tcttggactt ggagaagaat ggaggggagg agatgttgct agaactgttg 720 gaggaggaca gaaagtgaga tggcttaaga aagagatgga gaagtacgct gatagggagg 780 atatgattat tatgttcgtg gattcttacg atgtgattct tgctggatct ccaactgagc 840 ttttgaagaa attcgttcag tctggatcta ggcttctttt ctctgctgag tctttttgtt 900 ggccagaatg gggacttgct gagcaatatc cagaagtggg aactggaaag agattcctta 960 actctggagg attcattgga ttcgctacta ctattcacca gattgtgagg cagtggaagt 1020 acaaggatga cgatgatgat cagcttttct acactaggct ttaccttgat ccaggactta 1080 gggagaagtt gtctcttaac cttgatcaca agtctaggat tttccagaac cttaacggtg 1140 ctcttgatga ggttgtgctt aagttcgata ggaacagagt gaggattagg aacgtggctt 1200 acgatactct tcctattgtg gtgcatggaa acggaccaac aaaactccag cttaactacc 1260 ttggaaacta cgttccaaac ggatggactc cagaaggagg atgtggattc tgcaatcagg 1320 ataggagaac tcttccagga ggacaaccac caccaagagt tttccttgct gtgttcgttg 1380 aacagccaac tccattcctt ccaagattcc ttcagaggct tcttcttttg gattacccac 1440 cagatagggt gacacttttc cttcacaaca acgaggtttt ccacgagcca cacattgctg 1500 attcttggcc acagcttcag gatcatttct ctgctgtgaa gttggttggt ccagaagaag 1560 ctctttctcc aggagaagct agggatatgg ctatggattt gtgcaggcag gatccagagt 1620 gcgagttcta cttctctctt gatgctgatg ctgtgcttac taaccttcag actcttagga 1680 ttcttattga ggagaacagg aaagtgattg ctccaatgct ttctaggcac ggaaagttgt 1740 ggtctaattt ctggggtgct ctttctcctg atgagtacta cgctagatca gaggactacg 1800 tggagcttgt tcagagaaag agagtgggag tttggaacgt tccttatatt tctcaggctt 1860 acgtgattag gggagatact cttaggatgg agcttccaca gagggatgtt ttctctggat 1920 ctgatactga tccagatatg gctttctgca agtctttcag ggataaggga attttccttc 1980 acctttctaa ccagcatgag ttcggaagat tgcttgctac ttcaagatac gatactgagc 2040 accttcatcc tgatctttgg cagattttcg ataacccagt ggattggaag gagcagtaca 2100 ttcacgagaa ctactctagg gctcttgaag gagaaggaat tgtggagcaa ccatgcccag 2160 atgtttactg gttcccactt ctttctgagc aaatgtgcga tgagcttgtt gctgagatgg 2220 agcattacgg acaatggagt ggaggtagac atgaggattc taggcttgct ggaggatacg 2280 agaacgttcc aactgtggat attcacatga agcaagtggg atacgaggat caatggcttc 2340 agcttcttag gacttatgtg ggaccaatga ctgagtctct tttcccagga taccacacta 2400 aggctagggc tgttatgaac ttcgttgtga ggtatcgtcc agatgagcaa ccatctctta 2460 ggccacacca cgattcttct actttcactc ttaacgtggc tcttaaccac aagggacttg 2520 attatgaggg aggaggatgc cgtttcctta gatacgattg cgtgatttct tcaccaagaa 2580 agggatgggc tcttcttcat ccaggaaggc ttactcatta ccacgaggga cttccaacta 2640 cttggggaac tagatatatt atggtgtctt tcgtggatcc atgactgctt taatgagata 2700 tgcgagacgc ctatgatcgc atgatatttg ctttcaattc tgttgtgcac gttgtaaaaa 2760 acctgagcat gtgtagctca gatccttacc gccggtttcg gttcattcta atgaatatat 2820 cacccgttac tatcgtattt ttatgaataa tattctccgt tcaatttact gattgtccag 2880 aattcgcg 2888 <210> 39 <211> 2689 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the human Procollagen C-proteinase and flanking regions <400> 39 agatctatcg atgcatgcca tggtaccgcg ccatggctca attggctgca acatcaaggc 60 ctgaaagagt ttggccagat ggtgttattc ctttcgttat tggtggaaac tttactggat 120 ctcagagagc agtttttaga caagctatga gacattggga aaagcacact tgtgtgacat 180 tccttgaaag gactgatgaa gattcttata ttgtgttcac ataccgtcca tgtggatgct 240 gctcatatgt tggtagaagg ggaggaggtc cacaagcaat ttctattgga aaaaactgcg 300 ataagttcgg aattgtggtg catgaattgg gacatgttgt tggtttctgg cacgaacaca 360 caaggccaga tagggatagg cacgtgtcta ttgtgaggga aaacattcag ccaggtcaag 420 agtacaattt tcttaagatg gaacctcaag aggtggaatc tctcggagag acttacgact 480 tcgactccat catgcactac gcaaggaata ctttcagcag gggcatcttc ttggatacca 540 ttgtgcctaa gtacgaggtg aacggcgtta agccacctat tggtcaaagg actaggctct 600 ctaagggtga tattgcacag gctaggaagc tctacaaatg tccagcatgc ggagaaactc 660 ttcaggattc cactggcaac ttctcatctc cagagtaccc aaacggatac tctgctcata 720 tgcactgtgt ttggaggatc tcagtgactc ctggagagaa gatcatcctc aacttcactt 780 ccctcgatct ctatcgttct aggctctgtt ggtacgacta tgtggaagtg agagatggct 840 tctggagaaa ggctccactt agaggaaggt tctgcggatc taaacttcct gagccaatcg 900 tgtctactga ttccagattg tgggtggagt tcaggtcctc ttctaattgg gttggcaagg 960 gcttttttgc tgtgtacgag gctatttgtg gcggcgacgt gaaaaaggac tacggacata 1020 ttcaaagtcc aaattaccca gatgattacc gtccttcaaa agtgtgtatt tggaggattc 1080 aagtgagtga gggtttccat gttggattga cattccaatc tttcgaaatt gagagacacg 1140 attcatgcgc atacgattat ttggaagtga gagatggaca ctctgaatct tctacactta 1200 ttggaaggta ctgcggttat gagaaacctg atgatattaa gtctacttct agtaggttgt 1260 ggcttaaatt tgtgtcagat ggttctatta acaaggctgg tttcgcagtg aacttcttca 1320 aggaagtgga tgaatgctca agacctaaca gaggaggatg tgagcaaaga tgccttaaca 1380 ctttgggaag ttacaagtgt tcttgcgatc ctggatacga gttggctcct gataagagaa 1440 gatgcgaagc tgcttgcggt ggttttttga caaaattgaa cggatctatt acttctcctg 1500 gatggccaaa agagtaccca cctaataaga attgcatttg gcagcttgtt gcacctactc 1560 agtaccgtat ttcattgcaa ttcgattttt tcgagactga gggtaatgat gtgtgcaagt 1620 acgatttcgt ggaagtgaga tcaggtctta ctgctgatag taaattgcac ggaaagttct 1680 gcggatctga aaaaccagaa gtgattacat cacagtacaa caatatgagg gtggagttca 1740 aatctgataa tactgtttct aaaaaaggtt ttaaggcaca tttcttttct gataaggacg 1800 agtgctctaa agataatggt ggttgccagc aggattgcgt gaacacattc ggttcatatg 1860 agtgccaatg ccgtagtgga tttgttcttc acgataacaa acatgattgc aaagaggcag 1920 gttgcgatca caaggtgaca tctacttcag gtactatcac atctccaaac tggcctgata 1980 agtatccttc aaaaaaagaa tgtacatggg caatttcttc tacaccaggt catagggtta 2040 agttgacatt catggagatg gatattgaga gtcaaccaga gtgcgcttat gatcatcttg 2100 aggtgttcga tggaagggat gctaaggctc ctgttcttgg tagattctgt ggtagtaaaa 2160 agccagaacc agtgcttgca acaggatcta ggatgttcct tagattctac tctgataact 2220 cagttcagag gaaaggattc caagctagtc acgcaactga atgcggtgga caagttagag 2280 cagatgttaa gactaaggat ctttactcac acgcacagtt cggagataac aactaccctg 2340 gaggagttga ttgcgagtgg gttattgtgg ctgaagaggg atacggagtt gagcttgttt 2400 tccagacatt cgaggtggag gaggaaactg attgcggtta cgattatatg gaactttttg 2460 atggatacga tagtactgct ccaagacttg gaaggtattg tggtagtggt ccaccagaag 2520 aggtgtactc agctggagat agtgttcttg ttaagttcca cagtgatgat acaattacta 2580 agaagggatt ccatcttaga tatacttcaa ctaagtttca ggatactctt cattctagga 2640 agtaatgagc tcgcggccgc atccaagctt ctgcagacgc gtcgacgtc 2689 <210> 40 <211> 2912 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence containing the coding regions of the human Procollagen I N-proteinase and flanking regions <400> 40 gcgccatggc tcaattgagg agaagggcta ggagacacgc agctgatgat gattacaaca 60 ttgaagtttt gcttggtgtt gatgatagtg tggtgcaatt ccacggaaaa gagcatgttc 120 agaaatatct tttgacactt atgaatattg tgaacgaaat ctaccatgat gagtctttgg 180 gagcacacat taacgtggtt cttgtgagga ttattcttct ttcatacggt aaatctatgt 240 cacttattga gattggaaac ccttctcagt ctcttgagaa tgtgtgcaga tgggcatacc 300 ttcaacagaa gcctgatact ggacacgatg agtatcacga tcacgctatt ttccttacaa 360 ggcaggattt cggtccaagt ggaatgcaag gatatgctcc tgttactggt atgtgccacc 420 ctgttaggtc ttgtacactt aaccacgagg atggtttttc atctgctttc gtggtggctc 480 atgagacagg tcatgttttg ggaatggaac atgatggaca gggtaataga tgtggagatg 540 aagtgagact tggttcaatt atggctcctc ttgttcaagc tgcttttcat aggttccact 600 ggagtaggtg ttcacagcaa gagttgagta gataccttca ttcttacgat tgcttgcttg 660 atgatccatt tgctcatgat tggccagctt tgcctcaact tcctggattg cactactcta 720 tgaacgagca gtgcagattt gatttcggtc ttggttacat gatgtgcaca gctttcagga 780 ctttcgatcc atgcaaacag ttgtggtgtt cacacccaga taacccatat ttctgtaaaa 840 caaaaaaagg tccaccactt gatggtacta tgtgcgcacc tggaaagcac tgcttcaagg 900 gacactgcat ttggcttact cctgatattc ttaaaaggga tggatcatgg ggagcttggt 960 ctccattcgg aagttgctca agaacttgcg gaacaggtgt taagtttaga actaggcagt 1020 gcgataatcc acaccctgct aatggtggta gaacttgctc tggacttgct tacgattttc 1080 agttgtgttc taggcaagat tgccctgata gtcttgctga ttttagagaa gagcaatgta 1140 gacagtggga tctttacttt gagcacggcg acgctcagca ccactggctt ccacacgagc 1200 atagagatgc aaaagaaagg tgtcaccttt attgcgagag tagagagact ggagaggtgg 1260 tgtcaatgaa gagaatggtg cacgatggta caaggtgttc ttataaggat gcattctctt 1320 tgtgtgtgag gggagattgc aggaaagtgg gttgtgatgg agtgattgga tctagtaagc 1380 aagaagataa gtgcggagtg tgcggaggag ataactctca ttgcaaggtt gtgaaaggaa 1440 cttttacaag atcaccaaaa aaacacggtt acattaagat gttcgaaatt cctgctggag 1500 caaggcattt gcttattcag gaagtggatg caacatctca ccacttggca gtgaaaaacc 1560 ttgagactgg aaaattcatt ttgaacgagg agaacgatgt tgatgcatct agtaagactt 1620 tcattgcaat gggtgttgaa tgggagtata gggatgagga tggaagggaa acacttcaaa 1680 caatgggtcc tcttcatgga acaattactg tgttggtgat tccagtggga gatacaaggg 1740 tgtcattgac atacaagtat atgattcacg aggatagtct taacgttgat gataacaacg 1800 ttttggaaga agattctgtg gtttacgagt gggctcttaa gaaatggtca ccttgctcta 1860 agccatgtgg tggaggaagt cagttcacta agtatggttg taggaggagg cttgatcata 1920 agatggttca taggggattt tgcgcagcac ttagtaagcc aaaggcaatt aggagggctt 1980 gtaaccctca agaatgctca caaccagttt gggtgacagg agagtgggag ccatgttcac 2040 aaacatgcgg aagaactgga atgcaagtta gatcagttag atgcattcaa cctcttcatg 2100 ataacactac aagaagtgtg cacgcaaaac actgtaacga tgctaggcca gagagtagaa 2160 gagcttgctc tagggaactt tgccctggta gatggagggc aggaccttgg agtcagtgct 2220 ctgtgacatg tggaaacggt actcaggaaa gacctgttcc atgtagaact gctgatgata 2280 gtttcggaat ttgtcaggag gaaaggccag aaacagctag gacttgtaga cttggacctt 2340 gtcctaggaa tatttctgat cctagtaaaa aatcatacgt ggtgcaatgg ttgagtaggc 2400 cagatccaga ttcaccaatt aggaagattt cttcaaaagg acactgccag ggtgataaga 2460 gtattttctg cagaatggaa gttcttagta ggtactgttc tattccaggt tataacaaac 2520 tttcttgtaa gagttgcaac ttgtataaca atcttactaa cgtggagggt agaattgaac 2580 ctccaccagg aaagcacaac gatattgatg tgtttatgcc tactcttcct gtgccaacag 2640 ttgcaatgga agttagacct tctccatcta ctccacttga ggtgccactt aatgcatcaa 2700 gtactaacgc tactgaggat cacccagaga ctaacgcagt tgatgagcct tataagattc 2760 acggacttga ggatgaggtt cagccaccaa accttattcc taggaggcca agtccttacg 2820 aaaaaactag aaatcagagg attcaggagc ttattgatga gatgaggaaa aaggagatgc 2880 ttggaaagtt ctaatgagct cgcggccgca tc 2912 <210> 41 <211> 4467 <212> DNA <213> Homo sapiens <400> 41 atggctcacg ctcgtgttct cctcctcgct ctcgctgttt tggcaacagc tgctgtggct 60 gtggcttcta gttcttcttt tgctgattca aaccctatta gacctgttac tgatagagca 120 gcttccactt tggctcaatt gcaagaggag ggccaggttg agggccaaga tgaggatatc 180 cctccaatta catgcgtgca aaatggcttg cgttaccacg atagggatgt gtggaaacct 240 gaaccttgtc gtatctgtgt gtgtgataac ggcaaggtgc tctgcgatga tgttatctgc 300 gatgagacaa aaaattgccc tggcgctgaa gttcctgagg gcgagtgttg ccctgtgtgc 360 cctgatggtt ccgagtcccc aactgatcag gaaactactg gcgtggaggg cccaaaagga 420 gatactggtc cacgtggtcc taggggtcca gcaggtcctc caggtagaga tggtattcca 480 ggccagcctg gattgccagg accaccaggc ccacctggcc caccaggacc tcctggtctt 540 ggtggaaatt tcgctccaca actctcttat ggctatgatg agaagtcaac aggtggtatt 600 tccgttccag gtcctatggg accatccgga ccaagaggtc tcccaggtcc tccaggtgct 660 cctggacctc aaggctttca aggacctcca ggcgaaccag gagaaccagg cgcttctgga 720 ccaatgggcc caaggggacc acctggccca ccaggaaaaa atggcgatga tggcgaagct 780 ggaaagcctg gtcgtcctgg agagagaggt cctcctggcc cacagggtgc aagaggcttg 840 ccaggaactg ctggcttgcc tggaatgaag ggacataggg gcttctccgg cctcgatggc 900 gctaagggtg atgctggccc tgctggacca aagggcgagc caggttcccc tggagaaaac 960 ggtgctcctg gacaaatggg tcctcgtgga cttccaggag aaaggggtcg tccaggcgct 1020 ccaggaccag caggtgctag gggaaacgat ggtgcaacag gcgctgctgg ccctcctggc 1080 ccaactggtc ctgctggccc tccaggattc ccaggcgcag ttggagctaa aggagaagca 1140 ggaccacagg gccctagggg ttctgaagga cctcagggtg ttagaggtga accaggtcct 1200 ccaggcccag ctggagcagc tggtccagca ggaaatccag gtgctgatgg tcaacctgga 1260 gctaagggcg ctaatggcgc accaggtatc gcaggcgcac caggttttcc tggcgctaga 1320 ggcccaagtg gtcctcaagg accaggtgga ccaccaggtc caaaaggcaa ttctggcgaa 1380 cctggcgctc caggttctaa aggagatact ggtgctaaag gcgaaccagg acctgttggt 1440 gttcagggtc ctcctggtcc tgctggagaa gaaggaaaaa gaggtgctcg tggagaacca 1500 ggaccaactg gacttcctgg acctcctggt gaacgtggcg gacctggctc aaggggtttc 1560 cctggagctg atggagtggc aggtccaaaa ggccctgctg gagagagagg ttcaccaggt 1620 ccagctggtc ctaagggctc ccctggtgaa gcaggtagac caggcgaagc aggattgcca 1680 ggcgcaaagg gattgacagg ctctcctggt agtcctggcc cagatggaaa aacaggccca 1740 ccaggtccag caggacaaga tggacgtcca ggcccaccag gtcctcctgg agcaagggga 1800 caagctggcg ttatgggttt tccaggacct aaaggtgctg ctggagagcc aggaaaggca 1860 ggtgaaagag gagttcctgg tccaccagga gcagtgggtc ctgctggcaa agatggtgaa 1920 gctggagcac agggccctcc aggccctgct ggcccagctg gcgaacgtgg agaacaaggc 1980 ccagctggta gtccaggatt tcaaggattg cctggccctg ctggccctcc aggagaagca 2040 ggaaaacctg gagaacaagg agttcctggt gatttgggag cacctggacc ttcaggagca 2100 cgtggtgaaa gaggcttccc tggcgagagg ggtgttcaag gtccaccagg tccagcagga 2160 cctagaggtg ctaatggcgc tcctggcaac gatggagcaa aaggtgatgc tggtgctcct 2220 ggcgcacctg gaagtcaggg tgctcctgga ttgcaaggaa tgcctggaga gaggggtgct 2280 gctggcttgc caggcccaaa gggcgatagg ggtgatgctg gaccaaaagg tgctgatgga 2340 tccccaggaa aagatggagt tcgtggtctt actggcccaa tcggacctcc aggccctgct 2400 ggcgctccag gtgataaggg cgaaagtggc ccaagtggac ctgctggacc tactggtgct 2460 agaggtgcac ctggtgatag gggtgaacct ggaccacctg gtccagctgg ttttgctggt 2520 cctcctggag ctgatggaca acctggcgca aagggtgaac caggtgatgc tggcgcaaag 2580 ggagatgctg gtccacctgg acctgctggt ccagcaggcc cccctgggcc aatcggtaat 2640 gttggagcac caggtgctaa gggagctagg ggttccgctg gtccacctgg agcaacagga 2700 tttccaggcg ctgctggtag agttggccca ccaggcccat ccggaaacgc aggccctcct 2760 ggtcctccag gtcctgctgg caaggagggt ggcaaaggac caaggggcga aactggccct 2820 gctggtagac ctggcgaagt tggccctcct ggaccaccag gtccagcagg agaaaaaggt 2880 tccccaggag ctgatggccc agctggtgct ccaggaactc caggccctca aggtattgct 2940 ggacagagag gcgttgtggg actccctggt caaaggggag agagaggatt tccaggcttg 3000 ccaggaccta gtggagaacc tggaaaacaa ggcccatcag gcgctagtgg agagcgtgga 3060 cctcctggcc ctatgggacc tcctggattg gctggcccac ctggcgaatc aggtcgtgaa 3120 ggcgcaccag gcgcagaagg atcacctgga agagatggat cccctggtgc taaaggcgat 3180 cgtggagaaa ctggtccagc aggcccacca ggcgcaccag gtgcacctgg cgctccagga 3240 cctgtgggac cagctggaaa atccggagat aggggcgaga caggcccagc aggaccagct 3300 ggacctgttg gccctgctgg cgctcgtgga ccagcaggac ctcaaggacc aaggggagat 3360 aagggagaaa caggcgaaca aggcgatagg ggcattaagg gtcatagggg ttttagtggc 3420 ctccagggtc ctcctggccc acctggatca ccaggagaac agggaccatc tggtgcttcc 3480 ggcccagctg gtccaagagg acctccagga tcagctggtg cacctggaaa agatggtctt 3540 aacggtctcc caggaccaat cggccctcca ggacctagag gaagaacagg agatgctggc 3600 cctgttggcc ctccaggacc tcctggtcca ccaggtccac ctggtcctcc atcagctgga 3660 ttcgattttt catttcttcc acagccacca caagagaaag ctcacgatgg cggcagatat 3720 taccgtgctg atgatgctaa cgttgttagg gatagagatt tggaagtgga tacaactttg 3780 aaatccctct cccagcaaat tgaaaacatt agatctccag aaggttcacg taaaaaccca 3840 gctagaacat gtcgtgattt gaaaatgtgt cactccgatt ggaaaagtgg tgaatactgg 3900 attgatccaa atcagggctg taatctcgat gctatcaaag ttttctgtaa catggaaaca 3960 ggcgaaacat gcgtttatcc tactcaacct tccgtggctc agaaaaattg gtacatctca 4020 aaaaatccta aagataagag gcacgtttgg ttcggtgaaa gtatgactga tggatttcaa 4080 tttgagtacg gcggtcaagg tagtgatcca gctgatgtgg ctattcaact cacatttttg 4140 cgtcttatgt ccacagaggc atcacaaaac atcacttacc actgcaaaaa cagtgtggct 4200 tatatggatc aacaaacagg aaaccttaag aaggctcttc ttttgaaggg ctcaaacgag 4260 attgagatta gagcagaggg caactcaagg tttacttatt cagttactgt tgatggctgc 4320 acttcacata ctggcgcttg gggtaaaaca gttatcgagt ataagactac aaaaacatca 4380 agactcccaa tcattgatgt tgctcctctc gatgttggcg ctcctgatca agagttcggt 4440 tttgatgtgg gcccagtttg tttcctc 4467 <210> 42 <211> 4167 <212> DNA <213> Homo sapiens <400> 42 atggctcacg ctcgtgttct cctcctcgct ctcgctgttt tggcaacagc tgctgtggct 60 gtggcttcaa gttctagttt tgctgattcc aacccaattc gtccagttac tgatagagca 120 gcttccactt tggctcaatt gcttcaagaa gaaactgtga ggaagggccc tgctggcgat 180 aggggcccta ggggcgaaag gggtccacca ggacctccag gcagggatgg cgaagatggt 240 ccaactggcc ctcctggacc tcctggccct ccagggccac ccggcttggg cggaaacttc 300 gcagctcaat acgatggcaa gggtgttggt cttggtcctg gtcctatggg cttgatggga 360 cctagaggcc cacctggtgc tgctggtgct cctggaccac agggttttca gggaccagct 420 ggcgagccag gagagccagg ccaaacagga ccagctggtg caaggggacc tgctggacct 480 cctggaaaag ctggtgaaga tggtcaccca ggcaaaccag gacgtcctgg cgaaagaggt 540 gttgttggac cacaaggcgc taggggattt ccaggtacac ctggattgcc aggttttaag 600 ggcattcgtg gtcataacgg cctcgatgga ttgaagggac agcctggcgc acctggcgtt 660 aagggtgaac ctggagcacc aggtgaaaac ggtactcctg gccagactgg tgcaagagga 720 ctcccaggtg aaaggggtag agttggtgct cctggacctg ctggagctag gggtagtgat 780 ggtagtgttg gtcctgtggg ccctgctggt ccaatcggtt ccgctggccc acctggattc 840 ccaggcgctc caggacctaa aggagaaatc ggtgctgtgg gtaacgcagg tcctactggt 900 ccagcaggtc ctcgtggaga agtgggattg ccaggacttt ctggtccagt gggccctcca 960 ggcaaccctg gagctaacgg cttgacagga gctaaaggcg cagcaggact ccctggagtg 1020 gctggcgcac caggattgcc tggtccaagg ggtatcccag gccctgttgg cgcagctgga 1080 gctactggtg cacgtggact tgttggcgaa ccaggccctg ctggatcaaa aggcgagtct 1140 ggaaataagg gagaacctgg ttctgctgga cctcaaggtc ctcctggacc ttctggagaa 1200 gaaggaaaaa ggggaccaaa tggcgaggct ggatcagcag gtccaccagg accacctgga 1260 cttcgtggat cccctggtag tagaggactt ccaggcgctg atggtagagc aggcgttatg 1320 ggaccaccag gaagtagagg agcatccggt ccagcaggag ttaggggtcc taacggagat 1380 gctggtagac caggtgaacc aggtcttatg ggcccaaggg gcctcccagg tagtccagga 1440 aatatcggcc ctgctggaaa agaaggccct gttggacttc caggtattga tggacgtcct 1500 ggccctattg gcccagcagg tgcaagagga gaacctggca atattggatt tccaggacca 1560 aagggtccaa caggcgatcc tggaaaaaat ggagataagg gtcatgctgg attggcaggc 1620 gcaaggggcg ctcctggtcc agatggaaac aacggcgcac agggtccacc tggccctcag 1680 ggtgttcaag gcggaaaagg cgaacaaggc ccagctggac caccaggctt tcaaggcttg 1740 ccaggaccaa gtggtccagc aggtgaagtt ggcaagccag gcgagcgtgg acttcatggc 1800 gagtttggac tccctggacc agcaggacca aggggtgaaa gaggccctcc tggagagagt 1860 ggcgctgctg gaccaacagg cccaatcggt agtagaggtc ctagtggacc tccaggccca 1920 gatggaaata agggtgaacc aggagttgtg ggcgctgttg gaacagctgg tccttcagga 1980 ccatcaggac tcccaggcga gagaggcgct gctggcattc ctggaggaaa aggtgaaaaa 2040 ggcgaacctg gcctccgtgg cgaaatcgga aatcctggac gtgatggtgc tcgtggtgca 2100 cacggcgctg tgggcgctcc aggccctgct ggtgctactg gtgatagagg agaggctggc 2160 gcagctggcc cagcaggtcc tgctggccca aggggtagtc ctggtgaaag aggcgaagtt 2220 ggacctgctg gccctaacgg ctttgctggc cctgctggag cagcaggtca acctggcgct 2280 aaaggtgaaa ggggcggaaa gggcccaaaa ggtgaaaatg gcgttgtggg accaactggt 2340 ccagtgggcg cagctggacc tgctggtcca aatggaccac caggaccagc aggtagtaga 2400 ggagatggtg gacctccagg aatgacaggt tttccaggtg ctgctggtag aacaggacct 2460 cctggtccta gtggtatttc tggtccacca ggaccaccag gtcctgctgg aaaagaagga 2520 ttgaggggtc cacgtggtga tcaaggacca gtgggcagaa ctggtgaagt tggcgcagtg 2580 ggaccacctg gttttgctgg agaaaagggc ccttctggag aggcaggaac agctggtcct 2640 cctggtacac ctggacctca aggacttttg ggtgcacctg gtattctcgg attgccagga 2700 agtaggggcg aacgtggact tcctggcgtg gcaggagcag ttggagaacc tggccctctc 2760 ggaatcgcag gcccaccagg cgcaagagga ccaccaggag ctgttggatc accaggcgtg 2820 aatggtgcac ctggcgaggc tggtcgtgat ggaaacccag gaaatgatgg cccaccagga 2880 agagatggtc aacctggaca caaaggcgag aggggctacc caggaaatat tggcccagtt 2940 ggtgctgctg gcgcaccagg cccacacggt ccagttggac cagcaggaaa acacggtaat 3000 cgtggcgaaa caggcccttc aggcccagtg ggacctgctg gtgctgttgg cccaagagga 3060 ccatctggac ctcaaggcat tagaggcgat aagggagagc ctggcgaaaa aggacctaga 3120 ggcttgcctg gttttaaagg acacaacggt ctccaaggac ttccaggtat cgctggtcat 3180 catggagatc agggtgctcc tggatcagtg ggtccagcag gtcctagagg cccagcaggc 3240 ccttccggtc cagcaggaaa ggatggacgt actggccacc ctggaactgt gggccctgct 3300 ggaattagag gtcctcaagg tcatcagggc cctgctggcc ctccaggtcc accaggtcct 3360 ccaggcccac caggagtttc aggtggtggt tacgattttg gttacgatgg tgatttttac 3420 cgtgctgatc aacctagaag tgctccttct ctccgtccta aagattatga agttgatgct 3480 actttgaaat cacttaacaa ccagattgag actcttctca cacctgaggg atcaagaaag 3540 aatccagcac gtacatgccg tgatctcaga cttagtcacc cagagtggtc aagtggctat 3600 tattggattg atcctaatca gggttgtaca atggaggcta tcaaagttta ctgtgatttt 3660 ccaactggag agacatgtat tagggcacaa cctgagaaca ttccagctaa aaattggtat 3720 cgttcctcta aagataagaa acatgtttgg ctcggagaga ctattaacgc tggttctcag 3780 ttcgagtata atgttgaggg cgttacttct aaagagatgg caactcagct cgcttttatg 3840 agattgctcg ctaactacgc atcccaaaac atcacttatc actgcaaaaa ttccattgca 3900 tatatggatg aggagacagg aaatttgaag aaagcagtta ttctccaagg tagtaacgat 3960 gttgagcttg tggctgaggg aaatagtaga ttcacttaca cagttttggt ggatggatgc 4020 tcaaagaaaa ctaatgagtg gggcaagaca atcattgagt acaagacaaa taagccttct 4080 aggctcccat ttctcgatat tgcacctctt gatatcggag gagctgatca cgagtttttt 4140 gttgatatcg gacctgtttg ttttaag 4167

Claims (33)

As a double crosslinked dermal filler,
(a) plant-derived human collagen; And
(b) crosslinked hyaluronic acid
Wherein the plant-derived human collagen is crosslinked to crosslinked hyaluronic acid. The method of claim 1,
(a) the plant-derived human collagen comprises type 1 recombinant human collagen (rh collagen);
(b) the crosslinked hyaluronic acid comprises crosslinked hyaluronic acid and non-crosslinked hyaluronic acid;
(c) a combination thereof, a double crosslinked dermal filler. The method according to claim 1 or 2,
(a) the crosslinking agent for linking the crosslinked hyaluronic acid is different from the crosslinking agent for linking the plant-derived human collagen with crosslinked hyaluronic acid;
(b) the ratio of the crosslinked hyaluronic acid: the plant-derived human collagen is in the range of 6:1 to 1:6;
(c) the concentration of the crosslinked hyaluronic acid is in the range of 5 mg/ml to 50 mg/ml;
(d) a combination thereof, a double crosslinked dermal filler. The crosslinking agent for crosslinking hyaluronic acid and the crosslinking agent for crosslinking plant-derived human collagen are independently 1, 4-butanediol diglycidyl ether (BDDE), 1- [3-(dimethylamino)propyl]-3-ethylcarbodiimide metiodide (EDC), Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), a double crosslinked dermal filler selected from divinyl sulfone (DVS) or glutaraldehyde. A method for preparing a double crosslinked dermal filler comprising plant-derived human collagen crosslinked to crosslinked hyaluronic acid, the method comprising:
(a) crosslinking hyaluronic acid;
(b) neutralizing the crosslinked hyaluronic acid;
(c) neutralizing plant-derived human collagen;
(d) mixing the neutralized crosslinked hyaluronic acid with the neutralized plant-derived human collagen;
(e) adding a lower molecular weight hyaluronic acid (MW HA);
(f) crosslinking the mix of crosslinked hyaluronic acid and plant-derived human collagen; And
(g) dialysis of a double cross-linked cross-linked hyaluronic acid-plant-derived human collagen dermal filler. The method of claim 5,
(a) the plant-derived human collagen comprises type 1 recombinant human collagen (rh collagen);
(b) the crosslinking agent for linking the crosslinked hyaluronic acid of step (a) is different from the crosslinking agent for linking the plant-derived human collagen of step (f) with the crosslinked hyaluronic acid;
(c) a combination thereof. The method of claim 5 or 6,
(a) the ratio of the crosslinked hyaluronic acid: the plant-derived human collagen is in the range of 6:1 to 1:6;
(b) The crosslinking agent for crosslinking hyaluronic acid and the crosslinking agent for crosslinking plant-derived human collagen are independently 1, 4-butanediol diglycidyl ether (BDDE), 1-[3-(dimethylamino)propyl]-3 -Ethylcarbodiimide metiodide (EDC), Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), divinyl sulfone (DVS) or Is selected from glutaraldehyde;
(c) a combination thereof. A method of filling the tissue space under the epidermis, the method comprising:
(a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution,
(i) crosslinkable, plant-derived human collagen;
(ii) Hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof , Polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or modified derivatives thereof, carboxymethylcellulose or modified derivatives thereof , Crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; And
(iii) comprising a photoinitiator; And
(b) inducing polymerization by applying light to the surface of an epidermis superficial to the space. The method of claim 8, wherein the polymerizable solution components are introduced together or independently into the tissue space as a mixture at about the same place and at about the same time, and are independently introduced into the tissue space,
(a) crosslinkable, plant-derived human collagen and photoinitiator are introduced together and independently,
(b) the hyaluronic acid (HA) or a modified derivative thereof, the poly(vinyl alcohol) (PVA) or a modified derivative thereof, the polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or the Modified derivatives thereof, polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or modified derivatives thereof, carboxy The method, wherein methylcellulose or the modified derivative thereof, crystalline nanocellulose (CNC) or the modified derivative thereof, or a combination thereof is introduced into the tissue space independently at approximately the same time period. The method according to claim 8 or 9, further comprising the step of molding or sculpting the polymerizable solution or components of the polymerizable solution into a desired configuration within the tissue space, Wherein the step is simultaneous or subsequent to the step of applying light. 11. The method of claim 10, wherein the method is non-therapeutic and the shaping or shaping step reduces lines, folds, fine lines, wrinkles or scars, or a combination thereof. The method according to any one of claims 8 to 11,
(a) the crosslinkable, plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rh collagen);
(b) the hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), Modified derivatives of calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include methacrylated derivatives or thiolated derivatives;
(c) the hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), Calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) is crosslinked hyaluronic acid (HA), crosslinked poly(vinyl alcohol) (PVA), crosslinked polyethylene glycol (PEG), crosslinked oxidized cellulose (OC). ), crosslinked polymethylmethacrylate (PMMA) microspheres, crosslinked tricalcium phosphate (TCP), crosslinked calcium hydroxylapatite (CaHA), crosslinked carboxymethylcellulose, or crosslinked crystalline nanocellulose (CNC);
(d) a combination of (a) and (b), or a combination of (a) and (c). The method of claim 12, wherein when MA-rh collagen is selected, and when hyaluronic acid or a derivative thereof, or a crosslinked hyaluronic acid or a crosslinked derivative of hyaluronic acid is selected, HA or a derivative thereof or a crosslinked HA or a crosslinked HA derivative thereof The ratio of the derivative: MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1 :6, or 0:1, method. A method of filling the tissue space under the epidermis, the method comprising:
(a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution,
(i) crosslinkable, plant-derived human collagen; And
(ii) comprising a photoinitiator;
And
(b) inducing polymerization by applying light to the surface of an epidermis superficial to the space. 15. The method of claim 14, further comprising the step of shaping or shaping the polymerizable solution into a desired arrangement within the tissue space, the step being concurrent with or subsequent to the step of applying light. The method of claim 14 or 15, wherein the method is non-therapeutic, and the shaping or shaping step reduces wrinkles, folds, fine lines, wrinkles or scars, or a combination thereof. The method of any one of claims 14 to 16, wherein the crosslinkable, plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rh collagen). A method of filling a tissue space under the epidermis, the method comprising introducing a double crosslinked dermal filler into the tissue space, wherein the double crosslinked dermal filler comprises:
(a) plant-derived human collagen; And
(b) crosslinked hyaluronic acid (HA) or a modified crosslinked derivative thereof, crosslinked poly(vinyl alcohol) (PVA) or a modified crosslinked derivative thereof, crosslinked polyethylene glycol (PEG) or a modified crosslinked derivative thereof, crosslinked oxidized cellulose ( OC) or a modified crosslinked derivative thereof, a crosslinked polymethylmethacrylate (PMMA) microspheres or a modified crosslinked derivative thereof, a crosslinked tricalcium phosphate (TCP) or a modified crosslinked derivative thereof, a crosslinked calcium hydroxylapatite (CaHA) or A modified crosslinked derivative thereof, a crosslinked carboxymethylcellulose or a modified crosslinked derivative thereof, a crosslinked crystalline nanocellulose (CNC) or a modified crosslinked derivative thereof, or a combination thereof;
The plant-derived human collagen is crosslinked crosslinked hyaluronic acid (HA) or a modified crosslinked derivative thereof, crosslinked poly(vinyl alcohol) (PVA) or a modified crosslinked derivative thereof, crosslinked polyethylene glycol (PEG) or a modified crosslinked derivative thereof , Crosslinked oxidized cellulose (OC) or modified crosslinked derivative thereof, crosslinked polymethylmethacrylate (PMMA) microspheres or modified crosslinked derivative thereof, crosslinked tricalcium phosphate (TCP) or modified crosslinked derivative thereof, crosslinked calcium hydroxide A method of crosslinking to loxylapatite (CaHA) or a modified crosslinked derivative thereof, crosslinked carboxymethylcellulose or a modified crosslinked derivative thereof, crosslinked crystalline nanocellulose (CNC) or a modified crosslinked derivative thereof. The method of claim 18,
(a) the plant-derived human collagen is type 1 human recombinant collagen (rh collagen), or a MA or thiolated derivative thereof;
(b) the hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), Modified derivatives of calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include methacrylated derivatives or thiolated derivatives;
(c) a combination thereof. The method of claim 18 or 19, wherein when the crosslinked HA is selected, the ratio of the crosslinked HA: the plant-derived human collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1, method. 21. The method of any of claims 18-20, wherein the method is non-therapeutic, and the method reduces wrinkles, folds, fine lines, wrinkles or scars, or a combination thereof. As a polymerizable solution or a non-polymerizable solution for use in tissue strengthening,
(a) the polymerizable solution,
(i) crosslinkable, plant-derived human collagen,
(ii) Hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof , Polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or modified derivatives thereof, carboxymethylcellulose or modified derivatives thereof , Crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and
(iii) comprising a photoinitiator to induce polymerization prior to or concurrently with the application of visible light, or
(b) the non-polymerizable solution comprises a plant-derived human collagen, and a double-crosslinked dermal filler comprising crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE or crosslinked OC, wherein the plant-derived human collagen comprises the crosslinked Crosslinked with hyaluronic acid, the crosslinked PVA, the crosslinked PGE or the crosslinked OC;
The use includes injecting the polymerizable solution or non-polymerizable solution into the tissue space under the epidermis, followed by shaping or shaping the polymerizable solution or non-polymerizable solution into a desired configuration, such as wrinkles, folds, fine lines, A polymerizable solution or a non-polymerizable solution comprising the step of reducing wrinkles or scars. The method of claim 22,
(a) the crosslinkable, plant-derived human collagen is methacrylated or thiolated;
(b) the derivative of HA, PVA, PEG, PMMA, TCP, CaHA, or CNC includes a methacrylated derivative or a thiolated derivative;
(c) the derivatives of HA, PVA, PEG, PMMA, TCP, CaHA, or CNC include crosslinked derivatives;
(d) a polymerizable solution or a non-polymerizable solution, which is a combination thereof. The polymerizable solution or non-polymerizable solution of claim 22 or 23, wherein the tissue strengthening is required as a result of any medical or dental condition, wherein the dental condition comprises gum disease or gum replacement. . 25. The polymerizable or non-polymerizable solution of any one of claims 22 to 24, wherein the tissue strengthening reduces wrinkles, folds, fine lines, wrinkles or scars, or a combination thereof. A method for inducing a cellular growth promoting scaffold in a tissue space under the epidermis, the method comprising introducing a solution into the tissue space, the solution comprising:
(a) plant-derived human collagen; And
(b) at least one growth factor or a source thereof,
Wherein the method promotes healing or replacement of collagen-comprising tissue. The method of claim 26,
(a) the plant-derived collagen comprises type 1 recombinant human collagen (rh collagen);
(b) the source of the at least one growth factor comprises plasma or platelet-rich plasma;
(c) the collagen-comprising tissue comprises skin;
(d) any combination thereof. 28. The method of claim 26 or 27, wherein the rhcollagen comprises a methacrylate or thiol derivative thereof. The method of any one of claims 26 to 28, wherein the solution is
(a) Hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof , Polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or modified derivatives thereof, carboxymethylcellulose or modified derivatives thereof , Crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and
Photoinitiators to induce polymerization prior to or concurrently with the application of visible light; or
(b) a crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE or crosslinked OC, wherein the plant-derived human collagen is crosslinked to the crosslinked hyaluronic acid, the crosslinked PVA, the crosslinked PGE or the crosslinked OC. The method of any one of claims 26 to 29, wherein the method is non-therapeutic, and the cellular growth promoting scaffold is filled in a tissue space such that wrinkles, folds, fine lines, wrinkles or scars, or combinations thereof How to reduce it. A solution for use in inducing a cellular growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof, the use of which is injected into the tissue space under the skin And wherein the use is for promoting healing or replacement due to decomposition or injury of collagen-containing tissue. The method of claim 31,
(a) the source of the at least one growth factor comprises plasma or platelet-rich plasma;
(b) the plant-derived collagen comprises type 1 recombinant human collagen (rh collagen);
(c) the collagen-comprising tissue comprises skin;
(d) a solution, which is a combination thereof. The method of claim 31 or 32, wherein the solution for use is
(a) Hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof , Polymethylmethacrylate (PMMA) microspheres or modified derivatives thereof, tricalcium phosphate (TCP) or modified derivatives thereof, calcium hydroxylapatite (CaHA) or modified derivatives thereof, carboxymethylcellulose or modified derivatives thereof , Crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and
Photoinitiators to induce polymerization prior to or concurrently with the application of visible light; or
(b) a crosslinked hyaluronic acid, crosslinked PVA, crosslinked PGE or crosslinked OC, wherein the plant-derived human collagen is crosslinked to the crosslinked hyaluronic acid, the crosslinked PVA, the crosslinked PGE or the crosslinked OC.

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